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Recent genetic studies clearly indicate that variants in several lysosomal genes act as risk factors for idiopathic Parkinson's disease (PD). Variants in the co-activator of glucocerebrosidase gene (GBA) and the four active saposins (Sap A-D) which are encoded by the prosaposin gene (PSAP) are of particular interest; however, their genetic roles in PD are unknown. Whole-exome sequencing and Sanger sequencing were used to assess the genetic etiology of 400 autosomal dominant inherited PD (ADPD) and 300 sporadic PD (SPD) patients. Variants from public databases, including Genome Aggregation Database-East Asian (GnomAD_EAS) and Chinese Millionome Database (CMDB), were used as control groups. Burden analysis based on gene and domains level were performed to investigate the role of rare PSAP variants in PD. Six rare and likely pathogenic variants, located in the Sap A-D domains, were identified and accounted for 0.75% (3/400) of ADPD and 1.33% (4/300) of SPD in the Chinese population. Based on the gene or domain, burden analysis showed that damaging missense variants in SapC had statistical significance on the risk of developing PD. Interestingly, rs4747203, an intronic variant potentially linked to PSAP expression, was associated with reduced risk for PD (p = 8.6e-7 in GnomAD EAS and p = 0.002 in Chinese). Clinically, patients carrying the likely pathogenic variants presented typical PD motor symptoms and responded well to levodopa treatment. Six out of seven patients carrying the likely pathogenic variants of PSAP presented slow disease progression, and none of the patients developed cognitive impairment. Our study expands the spectrum of mutations associated with the risk of developing PD and enhances the understanding of the relationship of the clinical phenotype of PD with PSAP variants.

Knee osteoarthritis (OA) is an established risk factor for falls and balance impairment. This study investigated the incidence of falls, balance-related outcomes and risk factors for falls before and after primary total knee arthroplasty (TKA). Three hundred seventy-six OA patients scheduled to undergo TKA were included. Falls data within the preoperative, first postoperative and second postoperative years were collected, balance-related functions were assessed using the Assessment of Quality of Life (AQoL), WOMAC, Falls Efficacy Scale International (FES-I), Activities-specific Balance Confidence (ABC), knee extension strength, Berg Balance Scale (BBS) and Timed Up and Go (TUG) before surgery and 1 and 2 years after surgery. Compared with preoperative values, the incidence of falls significantly decreased (14.89%, 6.23% and 3.14% within the preoperative, first postoperative and second postoperative years, respectively) and the AQoL, WOMAC, FES-I, ABC, knee extension strength, BBS and TUG significantly improved after TKA. Logistic regression analysis revealed that Kellgren-Lawrence grade ≥ 3 of the contralateral knee was an independent risk factor for falls before and after TKA. Conclusively, primary TKA is associated with a reduced incidence of falls and improved balance-related functions, and the contralateral knee should be considered in the design of fall-prevention strategies in patients with OA.

Although peripheral nerve injury may result in a loss of function in innervated areas, the most effective method for nerve regeneration remains to be determined. The aim of the present study was to investigate the effect of control-released basic fibroblast growth factor (bFGF)-loaded poly-lactic-co-glycolic acid (PLGA) microspheres on sciatic nerve regeneration following injury in rats. bFGF-PLGA microspheres were prepared and their characteristics were evaluated. The sciatic nerve was segmentally resected to create a 10 mm defect in 36 Sprague Dawley (SD) rats and, following the anastomosis of the nerve ends with a silicone tube, bFGF-PLGA microspheres, free bFGF or PBS were injected into the tube (n=12 in each group). The outcome of nerve regeneration was evaluated using the sciatic function index (SFI), electrophysiological test and histological staining at 6 weeks and 12 weeks post-surgery. The bFGF-PLGA microspheres were successfully synthesized with an encapsulation efficiency of 66.43%. The recovery of SFI and electrophysiological values were significantly greater (P

Objective To investigate the expression of miRNA-140 in chondrocytes and synovial fluid of osteoarthritis (OA) patients, and explore the relationship between the miRNA-140 expression and OA severity.Methods This study enrolled 30 OA patients who underwent total knee arthroplasty for chondrocytes sampling and 30 OA patients who underwent intra-articular injection for synovial fluid sampling. All OA patients were grouped into mild [Kellgren and Lawrence (KL) grade 1-2], moderate (KL grade 3) and severe (KL grade 4), with 10 in each subgroups for each sampling purposes. 7 non-OA patients and 10 patients with knee injury were collected for cartilage and synovial fluid sampling respectively as control groups. Chondrocytes were isolated from the cartilage tissue and cultured in vitro. Quantitative real time PCR for miRNA-140 in chondrocytes and synovial fluid were performed, and the U6 snRNA was used as internal control. The expression difference of miRNA-140 among groups and correlation between the expression and the KL grade of OA were analysed using one-way ANOVA and Spearman test respectively. Results The expression of miRNA-140 in chondrocytes of knees in OA patients was reduced than that in normal knees, and the between-group difference was statistically significant (F=305.464, P

Summary Objective Disruptions of extracellular matrix (ECM) homeostasis are key events in the pathogenesis of osteoarthritis (OA). MicroRNA-140 (miRNA-140) is expressed specifically in cartilage and regulates ECM-degrading enzymes. Our objective in this study was to determine if intra-articular injection of miRNA-140 can attenuate OA progression in rats. Design miRNA-140 levels in human normal and OA cartilage derived chondrocytes and synovial fluid were assessed by polymerase chain reaction (PCR). After primary human chondrocytes were transfected with miRNA-140 mimic or inhibitor, PCR and western blotting were performed to quantify Collagen II, MMP-13, and ADAMTS-5 expression. An OA model was induced surgically in rats, and subsequently treated with one single intra-articular injection of miRNA-140 agomir. At 4, 8, and 12 weeks after surgery, OA progression were evaluated macroscopically, histologically, and immunohistochemically in these rats. Results miRNA-140 levels were significantly reduced in human OA cartilage derived chondrocytes and synovial fluid compared with normal chondrocytes and synovial fluid. Overexpressing miRNA-140 in primary human chondrocytes promoted Collagen II expression and inhibited MMP-13 and ADAMTS-5 expression. miRNA-140 levels in rat cartilage were significantly higher in the miRNA-140 agomir group than in the control group. Moreover, behavioural scores, chondrocyte numbers, cartilage thickness and Collagen II expression levels in cartilage were significantly higher, while pathological scores and MMP-13 and ADAMTS-5 expression levels were significantly lower in the miRNA-140 agomir group than in the control group. Conclusion Intra-articular injection of miRNA-140 can alleviate OA progression by modulating ECM homeostasis in rats, and may have potential as a new therapy for OA.

Background The aim of this study was to investigate the pancreas anatomy and surgical procedure for harvesting pancreas for islet isolation while performing pancreatectomy to induce diabetes in rhesus monkeys. Methods The necropsy was performed in three cadaveric monkeys. Two monkeys underwent the total pancreatectomy and four underwent partial pancreatectomy (70–75%). Results The greater omentum without ligament to transverse colon, the cystic artery arising from the proper hepatic artery and the branches supplying the paries posterior gastricus from the splenic artery were observed. For pancreatectomy, resected pancreas can be used for islet isolation. Diabetes was not induced in the monkeys undergoing partial pancreatectomy (70–75%). Conclusions Pancreas anatomy in rhesus monkeys is not the same as in human. Diabetes can be induced in rhesus monkeys by total but not partial pancreatectomy (70–75%). Resected pancreas can be used for islet isolation while performing pancreatectomy to induce diabetes.

The purpose of this study is to use diffusion tensor imaging (DTI) parameters to evaluate cerebral ischemia/reperfusion injury in the infarct core (IC) and ischemic penumbra (IP) in a rhesus transient middle cerebral artery occlusion (MCAO) model.Seven rhesus monkeys were used to construct the MCAO model. The temporal evolution of the relative apparent diffusion coefficient (rADC) and the relative fractional anisotropy (rFA) in the IC area, infarct growth area (IG), and reversible penumbra area (RP) were investigated.The rADC increased in the three areas in the early stage of reperfusion (1 hour after the reperfusion). However, the rate of rADC improvement was significantly slower in IG than in IC and RP. Different temporal evolutions of rFA were observed in the three areas in the following stage of reperfusion (3-24 hours after the reperfusion), which continued to decline in IG but slightly elevated in IC and RP.These findings suggest that the evolution of DTI parameters can help in the assessment of cerebral ischemia/reperfusion injury in the penumbra and predict the growth of the infarction area after stroke.

The high recurrence rate of hepatolithiasis, together with the high operative risk of hepatectomy for specifically located stones shows that an effective treatment for intrahepatic stones has not been settled upon. It is commonly accepted that a diseased biliary duct mucosa is a prerequisite for the development of intrahepatic stones, and that segmental biliary obstruction is able to induce hepatic atrophy, fibrosis, and "self-cut" the obstructed hepatic segment. Therefore, we previously put forward the hypothesis that performing deliberate chemical bile duct embolization (CBDE) to induce a segmental chemical hepatectomy might be the way of treating hepatolithiasis. In this study, we review the relative experimental basis for CBDE, preliminary report on its clinical use in 2 patients, and speculate on its future application. To completely embolize a diseased biliary duct, absolute ethanol or phenol is firstly used to ablate the biliary mucosa and eradicate biliary bacteria. Subsequently, cyanoacrylate or tissue adhesive glue is used to permanently fill the duct lumen, occupying the space where the stones would have formed. Our prior laboratory investigations and preliminary clinical treatments have confirmed that this combination of embolization agents could not only achieve the desired aim of preventing stone recurrence but could also lead to complete atrophy of the targeted hepatic segment, thereby achieving a chemical hepatectomy. In the future, CBDE is likely to help in resolving the problem of calculous recurrence and thereby reduce the incidence of surgical reintervention and endoscopic stone extractions, which are so frequently needed in patients with hepatolithiasis. Also chemical hepatectomy might provide a new less-invasive hepatectomy method, especially for the more difficult resections of the caudate or right posterior lobes.

Background and Aim: Chronic proliferative cholangitis (CPC) is currently considered as a pathological basis and major cause for the high recurrence rate of intrahepatic stones. Since CPC is a form of chronic proliferative disease, this study was designed to preliminarily investigate the inhibitory effect of proliferating cell nuclear antigen (PCNA) shRNA on the hyperplastic behavior and lithogenic potentiality of CPC. Methods: The rat model of CPC was given an intralumenal administration of 0.5 mL PCNA shRNA through a 20-gauge venous retained needle. PCNA shRNA-mediated effects on CPC-associated hyperplastic behavior and lithogenic potential were assessed by investigating histological changes, immunohistochemistry for Ki-67, biochemistry for β-glucuronidase, real-time polymerase chain reaction, and western blot analysis of PCNA, procollagen I, and mucin-3. Results: PCNA shRNA treatment could efficiently inhibit the mRNA and protein expressions of the proliferation-related gene, PCNA, and Ki-67, which efficiently inhibited the hyperplastic behavior of the biliary epithelium, submucosal gland, and collagen fibers in the diseased bile duct wall. This novel treatment could efficiently inhibit the formation of acidic mucus glands, the expression of mucin-3 mRNA, and the secretion of endogenous β-glucuronidase, thus effectively inhibiting the lithogenic potentiality of CPC. A further analysis revealed that PCNA shRNA-1 might display a more robust inhibitory effect on CPC-associated hyperplastic behavior and lithogenic potential than other gene sequences targeted in this study. Conclusions: PCNA shRNA-1 treatment could effectively inhibit the hyperplastic behavior and lithogenic potentiality of CPC, which might facilitate the prevention of stone recurrence and biliary restenosis.

This study evaluated the 5- to 19-year clinical and radiographic results of cementless acetabular revision. Between 1986 and 1998, 130 hips (125 patients) underwent cementless acetabular revisions. Ten patients were lost to follow-up; 6 patients died. One hundred nine patients (114 hips) were reviewed at a mean follow-up of 8.8 years. The mean Harris hip score improved from 62.1 to 90.7 at final follow-up. Two hips underwent repeat revision. Twenty-two hips developed cavitary osteolysis. Kaplan-Meier survivorship at 121 months was 98.2% with repeat revision for any reason as the end point and 89.5% with repeat revision or radiographic loosening as the end point. Cementless acetabular revision provides favorable clinical and radiographic results, and the initial disease and age may adversely affect the outcomes.

This study was designed to evaluate the midterm (or = 5 years) clinical and radiographic outcomes of the cementless total hip arthroplasty in 64 Chinese patients (72 hips), which revealed that the mean Harris hip score increased from an average of 44.0 points before operation to 92.4 points at the last follow-up. Excellent results were achieved in 60 hips (83%). No loosening of the components was observed radiographically. No revision of the femoral components was required. Only one focal area of pelvic osteolysis in 1 hip (1%), which requires a revision, and some small focal areas of femoral osteolysis in 12 hips (17%) were observed. The mean linear wear rate was 0.125 mm/y. The survival rate of the acetabular and femoral components for radiographic loosening was 100% (95% confidence interval, 0.93-1.0) and for revision was 98.61% (95% confidence interval, 0.95-1.0). This study indicated that the cementless total hip arthroplasty in patients with osteonecrosis of the femoral head has a satisfactory midterm clinical and radiographic outcomes, but the long-term effect should be further studied.

Total hip resurfacing arthroplasty (THRA) is being performed with increasing frequency for osteonecrosis of femoral head (ONFH). To evaluate femoral bone remodeling in ONFH after THRA and determine the impact of stem-neck angle (SNA) of inserted femoral component on bone remodeling, we monitored the changes in BMD in proximal femur in 23 patients with ONFH after surgery. Patients were divided into group A (SNA ≥ 5°) and group B (SNA

Similar to the heartbeat, gastric peristalsis is regulated by an electrical rhythm generated by a pacemaker. Thus, electrical dysrhythmia of gastric slow waves will inevitably affect gastric peristalsis and emptying. The recurrence of postoperative gastroparesis is thereby closely related to the abnormalities of electrical dysrhythmia and ectopic pacemakers, resulting in postoperatively persistent gastric motility disorders in some severe cases, despite the use of prokinetic and antiemetic drugs. Recent studies have demonstrated that gastric pacing, analogous to pacing the human heart, is an attractive and promising therapy that is both feasible and safe. Gastric pacing has been shown to be strikingly effective in normalizing gastric dysrhythmia, increasing the activity of the gastric slow wave and thereby prompting gastric dynamia and emptying. Furthermore, the long-term utilization of gastric pacing can (i) relieve patients from clinical symptoms, such as nausea and vomiting; (ii) release patients with severe postoperative gastroparesis from relying on prokinetic drugs and the jejunal feeding tube; (iii) return patients to normal oral nutritional intake and provide a more satisfactory nutritional status and most importantly; and (iv) give patients a better quality of life. Overall, research focused on gastric pacing has demonstrated excellent prospects for clinical application in the treatment of postoperative gastroparesis disorders, especially for those unresponsive to prokinetic drugs.

Background Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore, we investigated the liver expression profile of a highly inbred minipig line. Methods A cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme. Results Alignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5' terminal sequences and 140 complete 3' terminal sequences. Conclusion These newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation.

In the endothelium, ROS mainly derive from mitochondria, endothelial nitric oxide synthases and NADPH oxidases 4. Excessive ROS are a major cause of oxidative stress, the primary stimulus of vascular dysfunction and oxidative stress-related diseases. However, cellular evolution has made possible the development of adaptive antioxidant systems that scavenge excessive ROS, such as Nrf2/Keapl-ARE, PPAR-y, SIRT and FOXO, etc. Among them, the Nrf2/Keapl-ARE signaling pathway is perhaps the most prominent. What is more, there are the "crosstalk" among these antioxidant stress-related signaling pathways aim to alleviate oxidative stress injurys and promote cells survival. The understanding of the relationship between endothelial aging and oxidative stress may serve as a therapeutic clues in the treatment of oxidative stress-related diseases.

With the outstanding biocompatibility of hydroxyapatite (HA) and biodegradation of poly(D,L)lactide(PDLLA), and the expected good bio-mechanical compatibility, nano-HA / PDLLA (n-HA/PDLLA)composite has been paid great interests in hard tissue repair. One of the key factors affecting the potential of the composite is the degradation of the composite. That is what the mechanism of degradation in the composite is and if the degradation of the materials would induce the crack of the composite or a porous structure facile for tissue ingrowth would be formed. In this study, an n-HA/ PDLLA composite containing about 40% n-HA (wt%) was prepared and the degradation of the composite in bony tissue of rabbits and tissue response were studied by implanting composite rods and control HA rods into the femora of 16 New Zealand rabbits. After definite intervals, the histological analysis was completed by light microscopy and the degradation behavior was observed by scanning electron microscopy. The results suggested that a nano-HA/PDLLA composite was obtained and the materials showed good biocompatibility and osteoconductivity. The substantial degradation of the composite occurred at 8 weeks in vivo. After a longer period of implantation, the further degradation of the composite led to the formation of interconnected microporous and macroporous structure in the materials that might facilitate the tissue ingrowth in the composite.

The aim of this experiment is to investigate whether MSCs from Banna Minipig Inbred Line (BMI) could be immortalized by introducing SV40 large T antigen gene. MSCs were isolated from BMI and transfected with pSV3neo plasmid. Normal BMI-MSCs would apoptosis and senescence during proliferation while its population doubling (PD) number exceeded 20. However, SV40-transfected cells were immortal. As same as that of normal BMI-MSCs, transfected MSCs were positive for stem cell markers and negative for differentiated osteoblast specific marker. After cultured in osteogenesis supplement media, cbfa1 and calcium deposit on stimulated cells were enhanced obviously. There were no data to prove the tumorigenicity potential of the immortalized cells. Furthermore, histological analysis demonstrated that bone formation was initiated in the pores of HA/TCP implants loaded immortalized BMI-MSCs 7 weeks postimplantation. BMI-MSCs were immortalized by introducing SV40 large T antigen into the cells and still kept the stem cell characters and might be used as seeding cells for tissue engineering as well as stable test cells for biocompatibility of bone biomaterials.

Pig to human xenotransplantation is one of the possible ways to solve the problem of organ shortage, but the potential risk of xenozoonoses hinders the progress of xenotransplantation. Among pathogens that might cause xenozoonoses, porcine endogenous retrovirus (PERV) is undoubtedly the most noticeable. Current researches show that PERV exists in pigs' genomes and is able to express in multiple tissues and organs in pigs. Other experiments also show that PERV could successfully infect several human cell lines in vitro and could infect SCID mice through pig to mouse islet transplantation. The discussion and research on the risk of PERV is one of the hottest topics in current xenotransplantation, but the question of whether PERV will transmit to human through xenotransplantation has not been answered due to the lack of suitable animal models. More work should be done to evaluate the risk of xenozoonoses caused by PERV after pig to human xenotransplantation.

To investigate the optimal condition of bromodeoxyuridine (BrdU) labeling for bone marrow mesenchymal stem cells (BMSCs) in vitro and the feasibility of in vivo tracing of BrdU-labeling BMSCs.BMSCs were isolated from Wistar rats and were in vitro routinely cultured. The third passage BMSCs was used for identification of special surface antigens by immunohistochemical methods. The purified BMSCs were incubated with BrdU at different concentrations for different incubating time to investigate optimal BrdU concentration and incubating time for cell labeling. The cell labeling index of BrdU was calculated with immunohistochemical analysis. BMSCs labeled BrdU were injected into damaged gastric mucosa of rats by micro injector. The colonization of BMSCs labeled BrdU in gastric mucosa was viewed.After purification and proliferation, the primary cultured BMSCs were uniformly long spindle-shapped form and formed cell colony, which showed the characteristics of stem cell. Immunocytochemistry showed BMSCs were positive for CD44 and CD90, while negative for CD14, CD45. The labeling rate of BrdU increased with the labeling time lasting and reached its height at 48 h. After incubating 48 and 72 hours, the labeling rate of BrdU with a concentration of 10 micromol/L was higher than that of BrdU with a concentration of 5 micromol/L (P0.05) and similar with that of BrdU with a concentration of 15 micromol/L (P0.05). In addition, the BrdU labeling could be detected after five consecutive passages and the labeling time could keep 21 d. The pathological observation demonstrated that BrdU-labeled BMSCs could colonize the damaged gastric mucosa with normal morphologic characteristics during observation period.BrdU labeling might be a feasible method for dynamic observation of the migration, growth and differentiation of migrating BMSCs in colonizing sites.

The aim of this study was to investigate the pancreas anatomy and surgical procedure for harvesting pancreas for islet isolation while performing pancreatectomy to induce diabetes in rhesus monkeys.The necropsy was performed in three cadaveric monkeys. Two monkeys underwent the total pancreatectomy and four underwent partial pancreatectomy (70-75%).The greater omentum without ligament to transverse colon, the cystic artery arising from the proper hepatic artery and the branches supplying the paries posterior gastricus from the splenic artery were observed. For pancreatectomy, resected pancreas can be used for islet isolation. Diabetes was not induced in the monkeys undergoing partial pancreatectomy (70-75%).Pancreas anatomy in rhesus monkeys is not the same as in human. Diabetes can be induced in rhesus monkeys by total but not partial pancreatectomy (70-75%). Resected pancreas can be used for islet isolation while performing pancreatectomy to induce diabetes.

To investigate the effect of immunosuppression agent Everolimus on the viability and function of insuloma cells (INS-1) and pancreatic islet cells cultured in vitro.INS-1 cells and islets were treated with a series of concentrations of immunosuppression agents (Everolimus, Cyclosporin A, Sirolimus and Mycophenolate Mofetil). The viability of INS-1 cells and rat pancreatic islets were determined with MTT and the function of INS-1 cells and rat pancreatic islets was evaluated with Glucose-stimulated insulin secretion assay.Above the clinical blood concentration, the inhibition rate of islet cell proliferation in the high concentration group of Everolimus and Sirolimus was significantly lower than that of Cyclosporin A and Mycophenolate Mofetil group (P0.05); Everolimus in the blood drug level, like other immunosuppressive agents, can inhibit the function of insulin secretion, and the stimulation index of each group was no significant difference.Compared to Mycophenolate Mofetil and Cyclosporin A, Everilimus and Sirolimus demonstrate lower toxicity effect on INS-1 cells and rat pancreatic islets in vitro and Everolimus is expected as a new type of immunosuppressive agent used in clinical islet transplantation.

High stone recurrence and biliary restenosis rates in hepatolithiasis patients have been confirmed to be closely related to postoperatively-remnant chronic proliferative cholangitis (CPC), but effective management strategies have not yet been developed. Since CPC is a type of hyperplastic disease, this study was designed to investigate inhibitory effectiveness of cdc2 k ShRNA on hyperplastic behavior and lithogenic potentiality of CPC.0.5 ml of P-cdc2 shRNA was injected transpapillarily into the bile duct lumen in a rat model of cholangitis. Then, the effects of cdc2 k ShRNA on CPC were evaluated by histology, immunohistochemistry, RT-PCR, Western blot, biochemistry and enzymatic histochemistry for cdc2 k, PCNA, Ki-67, Procollagen III, Mucin 5AC, beta-glucuronidase and hydroxyproline.cdc2 k shRNA-3 treatment could efficiently inhibit hyperplasia of biliary epithelium, submucosal gland, and collagen fiber by inhibiting mRNA and protein expressions of the proliferation-related gene, cdc2 k, PCNA and Ki-67, thus holding the promise to control or reverse CPC and its secondary biliary stricture. Also of note, this novel treatment may decrease the lithogenic potential of CPC via inhibition of endogenous beta-glucuronidase and Mucin 5AC expression, hereby facilitating the prevention of stone recurrence.cdc2 k shRNA-3 treatment could effectively inhibit the hyperplastic behavior and lithogenic potentiality of CPC, which might help to prevent the biliary restenosis and stone recurrence.

To explore a suitable method for isolation, purification and multiplication of bone marrow mesenchymal stem cells (BMSCs).Density gradient centrifugation and adherence separation methods were applied for isolation of BMSCs from Wistar rats. The cells were cultured and proliferated in culture medium containing calf serum (CS), fetal bovine serum (FBS), free of serum or different volume fraction of FBS. The characteristic and the morphology of BMSCs were observed under inverted microscope every day. The growth curves were draw and the surface antigen of BMSCs were detected by immunocytochemistry technique. The microstructure was observed by transmission electron microscope (TEM).The pure primary cells can be procured by density gradient centrifugation. But the primary cells cultured by adherence separation methods demonstrated higher cytoactive, more rapid proliferation, earlier colony confluence and shorter time for passage than that cultured by density gradient centrifugation method. The cells by adherence separation methods were essentially purified at passage 4. Both CS and FBS can promote the growth and proliferation of BMSCs, but the colony forming efficacy of cells (46.50%) cultured in medium containing 0.12 volume fraction FBS was the highest. The cells surface markers CD44, CD90 were positive and CD14, CD45 were negative. BMSCs were observed by TEM and possessed the characteristic of stem cells. Conclusion BMSCs with high quality and activity can be obtained with adherence separation by suitable method and culture conditions. L-DMEM medium containing 0.12 volume fraction FBS showed more profitable for the growth and proliferation of BMSCs.

To compare the effects of enteral nutrition (EN) and parenteral nutrition (PN) on gut mucosal barrier function and intestinal epithelial tight junctions in rats after surgical stress.Fifty SD rats with surgical trauma were randomly divided into three groups: total parenteral nutrition (TPN) group and enteral nutrition (EN) group with isocaloric and isonitrogenous nutrition and placebo group. Nutrients were administered via the neck vein and needle jejunostomy for the TPN group. The homogenated tissues of liver, lung, and mesenteric lymph nodes were cultured to determine the bacterial translocations rates. The transmembrane binding proteins (occludin) were measured with immunohistochemistry. The ultrastructure and morphology of intestinal epithelial tight junctions were observed by electron microscope. The feces in cecum were cultured for anaerobic bacterial growth analyses.The EN group had more lactobacteria and bifydobacteria than the TPN group, but not statistical significant. The EN group had greater expression of occludin in the intestines than the TPN group. Furthermore, the intestinal epithelial tight junction and microvilli of the EN group were more intact compared with those of the TPN group. The bacterial translocations rates of liver, lung and mesenteric lymph nodes were significantly lower in the EN group than in the TPN group.Neither EN nor standard PN maintains intestinal membrane barriers. But EN increases the expression of transmember binding proteins, maintains the gut epithelial tight junction, improves intestinal mucosal barrier and reduces gut bacterial translocation.

Recently, high stone recurrence and biliary restenosis rates in hepatolithiasis patients have been confirmed to be closely related to chronic proliferative cholangitis (CPC). However, the effective management of CPC has not yet been established.A vicious cycle exists between the presence of intrahepatic calculi and CPC: both the stone itself and secondary biliary infection can stimulate persistent hyperplasia in the biliary duct wall, leading to the occurrence of CPC and biliary stricture. The recurrent attacks of CPC will, in turn, facilitate new stone formation via mucoglycoprotein production, or induced biliary stricture and cholestasis. Thus, even when the stone is completely removed and the biliary tract stenosis is corrected, residual CPC will persist and progress, with an underlying risk for postoperative stone recurrence and biliary tract restenosis. Therefore, the perfect hepatolithiasis treatment would target stone removal and correction of the biliary tract stricture, as well as control of postoperative residual CPC. In fact, CPC, the management of which has been traditionally ignored, is the key to breaking this vicious cycle.Overall, the subsequent treatment of residual CPC after operation or choledochoscopic lithotomy would be helpful to decrease postoperative stone recurrence and the rate of biliary restenosis. Adding such treatment would reduce the incidence of surgical reintervention and choledochoscopic lithotomy, and it would also improve the postoperative hepatolithiasis outlook.

To investigate the effects of ecoimmunonutrition supplement on intestinal microecology, epithelial tight junctions, and barrier function in rats with surgical stress.Seventy SD rats after surgical trauma were randomly divided into four groups:(1) placebo group,(2)total parenteral nutrition(TPN) group,(3)enteral nutrition(EN) group and (4)ecoimmunonutrition (EEN)group respectively. Rats received isocaloric and isonitrogenous nutrition. Nutrients were administered via the neck vein and the needle jejunostomy for five days. The homogenated tissues of liver, lung, and mesenteric lymph nodes were cultured to determine the bacterial translocation rate. The transmembrane binding proteins(occludin) was measured by immunohistochemistry. The ultrastructure and morphology of intestinal epithelial tight junctions in the intestine were observed by electron microscope. The feces in cecum was cultured for anaerobic bacterial growth and analysed.The amounts of lactobacteria and bifidobacteria in EEN group were significantly higher than those in TPN group(P0.05). The expression levels of occludin in the intestine was significantly higher in EEN group than that in TPN and EN group. Furthermore, the intestinal epithelial tight junction and microvilli of EEN group were more intact compared with those of TPN group. The bacterial translocation rates of liver, lung and mesenteric lymph nodes were significantly lower in EEN and EN group than those in TPN group(P0.05).Application of ecoimmunonutrition can protect intestinal mucosal barrier in rats with operative stress, increase the expression of occludin, maintain the gut epithelial tight junction, and eliminate gut bacterial translocation.

To investigate the potential of small interfering RNA (siRNA) against human peroxisome proliferator activated receptor gamma (PPARgamma) to suppress the adipogenic effect of alcohol on human bone marrow-derived mesenchymal cells (hBMSCs) and increase osteogenesis.hBMSCs collected from joint replacement surgery were cultured, passaged, and transfected with PPARgamma-siRNA by using liposomal-based strategy. Then the cells were maintained in culture and treated with alcohol 50 mmol/L and dexamethasone, an osteogenic inducer, for 24 days. Oil red O staining was used to observe the fat drops in the cells so as to count the number of adipocytes. Histochemistry was performed to detect the protein expression of alkaline phosphatase, osteocalcium, and type 1 collagen on days 24 and 28 after treatment. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of osteoblast-specific transcription factor-2 (Osf2)/core-binding factor alpha subunit 1 (Cbfa1) on day 10.The adipocyte number of the PPARgamma-siRNA group was significantly lower than those of the alcohol-induced group and the controls (adipogenic group, liposomal group, and negative siRNA group) (all P0.05). The Calcium nodule formation rate of the PPARgamma-siRNA group was significantly higher than alcohol-induced group and the controls (adipogenic group, liposomal group, and negative siRNA group) (all P0.05). PCR and Western blotting showed significantly higher mRNA and protein expression of the human adipocyte-specific markers:Cbfa1, ALP, type 1 collagen, and osteocalcin, than the other groups.The adipogenic effect of alcohol on hBMSCs can be inhibited with a concomitant increase in osteogenesis by using siRNA technique to specific targeted human PPARgamma gene in vitro cell culture systems. PPARgamma-siRNA strategy may be a useful tool for studying the mechanisms of alcohol-induced osteoporosis and provides theoretical basis for its genetic therapy.

To evaluate the changes of periprosthetic bone mineral density in femur after hip resurfacing arthroplasty.From July 2002 to June 2005, a comparative study was carried out on 52 hips in 52 patients. Twenty-six patients (26 hips) who underwent Birmingham hip resurfacing arthroplasty (group BHR), and 26 patients (26 hips) who performed cementless total hip arthroplasty with Versys System stem (group THA). The periprosthetic bone mineral density of the femur was measured through dual energy X-ray absorptiometry of the Gruen zones at pre-operation, post-operation 3, 6, 12 and 24 months in patients from both group BHR and group THA. The bone mineral density of femoral neck in group BHR was measured too. Changes of bone mineral density ratio in proximal femur between pre-operation and post-operation were compared and analyzed.The mean ratio of bone mineral density of the proximal femur in group BHR reduced by 5.8%, 4.9%, 2.6% and 0.4%, in group THA reduced by 7.0%, 10.6%, 1.0% and 4.1% at 3, 6, 12 and 24 months respectively. In group BHR, the mean ratio of bone mineral density in range of interest 1 decreased to 89.7% at 6 months and increased to 103.8% at 24 months, in range of interest 7 decreased to 95.1% at 6 months and increased to 103.7% at 24 months. In group THA, the mean ratio of bone mineral density in range of interest 1 decreased to 90.8% at 6 months, 94.4% at 24 months and in range of interest 7 decreased to 94.2% at 3 months, 96.7% at 24 months. In group BHR, the bone mineral density of femoral neck was restored to the pre-operation level at 6 months. The bone mineral density in superior-lateral zone of femoral neck decreased to 97.1% at 3 months and increased to 107.4% at 24 months respectively. The bone mineral density in inferior-medial zone of femoral neck increased to 117.9% at 24 months.The bone stock of proximal femur can be well preserved and recovered quickly after hip resurfacing arthroplasty.

To discuss the method of reconstruction of acetabular bone defect with wire mesh, impaction bone-grafting and a cemented cup in acetabular component revision.21 hips in 21 patients, aged 50.1 (31 - 64), 2 hips being of the acetabular defect type I B, 1 hip of type II A, 4 hips of type II B, and 14 hips of type III according to the American Academy of Orthopaedic Surgeons (AAOS) grading system underwent reconstruction with wire mesh, impacted bone grafts and cemented polyethylene acetabular component, and then were followed up for 47 months (36 - 60 months).The mean Harris hip score improved from the preoperative 55.7 points to 92.9 points at the last time of follow-up. Radiographic incorporation between host the bone and allograft was achieved 11.4 months after the operation on average. The mean change of inclination of acetabular components was 2.2 degrees , in which one acetabular component developed a change of 15.5 degrees . The inclination of acetabular components increased by 1.7 degrees on average 3 months after the operation and 2.0 degrees 6 months after the surgery. The acetabular cup migrated medially and superiorly by 3.93 mm and 4.41 mm respectively, peaking in the sixth month after the operation. One hip developed heterotopic ossification (Brooker grade I). One hip received repeat revision because of the aseptic loosening of the acetabular component 25 months after the revision surgery.The changes of the position of acetabular cup mainly occur within six months after the reconstruction of severe acetabular bone defect with wire mesh, impacted bone graft and cemented polyethylene acetabular component. To prevent the displacement of the acetabular cup, it is necessary to keep initial stability of the acetabular cups within six months after the index surgery.

There is a direct relationship between mechanical stress and the progressive collapse of the necrotic region in osteonecrosis of the femoral head. The titanium implant combined with autogenous bone graft and recombinant human bone morphogenetic protein (rhBMP)-2 to repair the defect and prevent collapse of the femoral head was investigated. The femoral head defects were made by the trapdoor procedure and then the defects were filled, respectively, with the titanium implant combined with autogenous bone graft and rhBMP-2, with autogenous bone graft and rhBMP-2, and with autogenous bone graft alone. Roentgenographic and histological examinations were performed at various times postoperatively. The defects were repaired completely and the titanium implant was integrated with the surrounding bone tissues. The defects healed faster than did without rhBMP-2. No trapdoor cartilage collapsed and joint space narrowed. The titanium implant combined with autogenous bone graft and rhBMP-2 can enhance the repairing procedure and prevent the collapse of the femoral head.

Porcine liver performing efficient physiological functions in the human body is prerequisite for successful liver xenotransplantation. However, the protein differences between pig and human remain largely unexplored. Therefore, we investigated the liver expression profile of a highly inbred minipig line.A cDNA library was constructed from liver tissue of an inbred Banna minipig. Two hundred randomly selected clones were sequenced then analysed by BLAST programme.Alignments of the sequences showed 44% encoded previously known porcine genes. Among the 56% unknown genes, sequences of 72 clones had high similarities with known genes of other species and the similarities to human were mostly above 0.80. The other 40 clones showing no similarity to genes in National Centre for Biotechnology Information are newly discovered, expressed sequence tags specific to liver of inbred Banna minipig. Twenty-two of the 200 clones had full length encoding regions, 38 complete 5' terminal sequences and 140 complete 3' terminal sequences.These newly discovered expression sequences may be an important resource for research involving physiological characteristics and medical usage of inbred pigs and contribute to matching studies in xenotransplantation.

This study evaluated the 5- to 19-year clinical and radiographic results of cementless acetabular revision. Between 1986 and 1998, 130 hips (125 patients) underwent cementless acetabular revisions. Ten patients were lost to follow-up; 6 patients died. One hundred nine patients (114 hips) were reviewed at a mean follow-up of 8.8 years. The mean Harris hip score improved from 62.1 to 90.7 at final follow-up. Two hips underwent repeat revision. Twenty-two hips developed cavitary osteolysis. Kaplan-Meier survivorship at 121 months was 98.2% with repeat revision for any reason as the end point and 89.5% with repeat revision or radiographic loosening as the end point. Cementless acetabular revision provides favorable clinical and radiographic results, and the initial disease and age may adversely affect the outcomes.

The high operative risk of hepatectomy for specially located intrahepatic stones is still a problem to be solved. This study was undertaken to investigate the feasibility and effectiveness of chemical bile duct embolization for chemical hepatectomy.Oxybenzene or absolute ethanol plus N-butyl-cyanoacrylate was employed for embolization. The feasibility, effectiveness and mechanism of chemical hepatectomy were preliminarily analyzed histologically or by Fas, TIMP-1, TGF-beta(1), and collagen I.Oxybenzene plus cyanonacrylate can preferably destroy and embolize the intrahepatic biliary duct, leading to the disappearance of hepatocytes in the periphery of embolized lobe and the achievement of effective chemical hepatectomy. The expressions of Fas, TIMP-1 and TGF-beta(1) in oxybenzene embolism group (88.90 +/- 38.10, 619.43 +/- 183.42, 185.22 +/- 70.39) and ethanol embolism group (72.39 +/- 29.51, 407.55 +/- 134.74, 163.56 +/- 51.75) were higher than those of biliary duct-ligated group (26.31 +/- 12.07, 195.31 +/- 107.67, 74.84 +/- 40.73) (P0.05). The collagen I-positive percentage in the oxybenzene embolism group was also greater than that of the ethanol embolism group (33.97 +/- 12.51% vs. 20.67 +/- 8.09%, P0.05).The effect of chemical hepatectomy may be achieved by chemical bile duct embolization.

To develop an HPLC method for the determination of 6-methylmercaptopurine concentration and then numerate the thiopurine methyltransferase (TPMT) activity in erythrocyte via formula and analysis of the TPMT activity difference between human and pig.The chromatographic apparatus SHIMADZU GC-2010C was used. The stationary phase was a Symmetry C18 reverse-phase column (150 mm x 3.9 mm, 5 microm). The mobile phase was 0.1% acetic acid-methanol and the flow rate 1.0 mL/min. The samples were extracted by ethyl acetate and injected automatically. They were measured at UV 280 nm. 2-amino-6-methyl-mercaptopurine was used as the internal standard.The retention times for 6-MP, AMMP and 6-MMP were 4.4 min, 7.7 min and 9.3 min respectively. The calibration curves were linear over the range of 6.25-100 nmol/mL. the methodology recovery was 98.08%-100.05%. The extraction recovery of 6-MMP was 88.1%-92.4%. The within-group RSD 1.0%-1.5% and inter-group RSD 1.0%-4.4%. The levels of TPMT activity of human, pig and Banna Minipig Inbred Line (BMI) were (17.45 +/- 3.62) U, (7.65 +/- 1.35) U and (8.73 +/- 1.55) U respectively.The method is suitable for determination of TPMT activity in erythrocyte of different patients and species in that of clinical medicine and scientific research. TPMT activity of pig and BMI is obviously lower than human's.

Background: The high recurrence rate of hepatolithiasis is still a problem to be solved. The purpose of the present study was therefore to carry out a preliminary study of the practical value of chemical bile duct embolization (CBDE) to treat hepatolithiasis in rabbits. Methods: Chemical bile duct embolization was performed with phenol or absolute ethanol along with N-butyl-cyanoacrylate. The feasibility and effectiveness of CBDE for chemical hepatectomy was assessed by investigating histological changes, biochemistry for hydroxyproline and in situ hybridization for collagen I. Results: Histologically, the mucosal epithelia of the embolized bile ducts were entirely replaced by collagen fibers, thus effectively eradicating chronic proliferative cholangitis. Also of note, the diseased biliary duct lumens were completely filled with N-butyl-cyanoacrylate, thus effectively preventing calculus formation. The hepatocytes also disappeared completely in the periphery of the embolized lobe, demonstrating that the desired effects of chemical hepatectomy were achieved through CBDE. In a further comparison of embolizing agents, the phenol-cyanoacrylate embolized livers and bile ducts had a higher level of hydroxyproline and collagen I than those embolized with ethanol plus cyanoacrylate. Conclusion: Chemical bile duct embolization is a promising approach to prevent the recurrence of hepatolithiasis and to achieve the effect of chemical hepatectomy.

A Bacillus strain, denoted as PY-1, was isolated from the vascular bundle of cotton. Biochemical, physiological and 16S rDNA sequence analysis proved that it should belong to Bacillus subtilis. The PY-1 strain showed strong ability against many common plant fungal pathogens in vitro. The antibiotics produced by this strain were stable in neutral and basic conditions, and not sensitive to high temperature. From the culture broth of PY-1 strain, five antifungal compounds were isolated by acidic precipitation, methanol extraction, gel filtration and reverse-phase HPLC. Advanced identification was performed by mass spectrometry and nuclear magnetic resonance spectroscopy. These five antifungal compounds were proved to be the isomers of iturin A: A2, A3, A4, A6 and A7. In fast atom bombardment mass spectrometry/mass spectrometry collision-induced dissociation spectra, fragmentation ions from two prior linear acylium ions were observed, and the prior ion, Tyr-Asn-Gln-Pro-Asn-Ser-betaAA-Asn-CO+, was first reported.

To assess the infectivity of porcine endogenous retrovirus (PERV) via in vitro infection of human embryonic kidney cell line HEK-293.PERV particles were detected by immunoelectron microscopy. PERV DNA and mRNA were studied in HEK-293 24 hours after the infection using polymerase chain reaction and reverse transcriptase-PCR respectively. The PERV types were also analyzed. PERV-gag protein was observed by confocal microscopy.Retroviral particles were round under electron microscope. PERV-gag pol gene and gag protein were detected and expressed in the infected HEK-293 cells. The types of PERV were PERV-A and PERV-B. PERV-gag protein was also identified in the cytoplasm of infected cells by confocal microscopy.PERV is able to infect HEK-293 cell line in vitro; types of PERV-gag protein is also expressed as a result. Further studies are thus necessary in order to evaluate the possibility of xenozoonoses in pig-to-human xenotransplantation.

The high recurrence of hepatolithiasis, together with the high operative risk of hepatectomy for specially located stones, has not been settled effectively to date. Thus, this study was designed to investigate the feasibility of applying chemical biliary duct embolization (CBDE) to chemical hepatectomy in rats. As revealed in our results, the intrahepatic biliary ducts could be partially or completely occluded by both phenol and absolute ethanol. In addition, the embolization effect was greatly enhanced by further using cyanoacrylate. Also noteworthy is that CBDE resulted in massive death of hepatocytes, which were replaced by proliferated bile ductules and collagen. More importantly, the hepatocytes disappeared completely in the periphery of the embolized lobe where chemical hepatectomy was achieved. As for the comparison of embolic agents, the combination of phenol and cyanoacrylate exhibited even better fibrogenic effects than the combination of ethanol and cyanoacrylate. In conclusion, CBDE might be a promising approach for achieving the effects of chemical hepatectomy. The combination of phenol and cyanoacrylate potentially acted as a more effective agent for biliary duct embolization.

To investigate the antibacterial activity and the cytotoxicity of polypeptide dhvar4.Solid-phase synthesized dhvar4 was manufactured by MeiLian Biotech Co. Ltd.; we evaluated its antibacterial effect, using the growth curve of bacteria after the bacteria being treated with different concentration of dhvar4. The cytotoxicity of dhvar4 on L-02, ECV-304, 293, 3T3 cells was investigated by MTT assay, and human erythrocytes were subimitted to hemolytic assay.dhvar4 inhibited the growth of Enterococcus faecium 36418 and Staphylococcus aureus BAA42 only at the concentration of 4 microg/ml. The viability of mammalian cells remained 68%-92% even when the concentration of dhvar4 reached 150 microg/ml. The LD50 (median lethal dose) was determined to be higher than 150 microg/ml. And very low hemolytic activity of dhvar4 was detected even at the highest protein concentration tested (180 microg/ml).dhvar4 possesses antibacterial activity and has little cytotoxicity to mammalian cells. These results suggest that dhvar4 has great potential in the development of

To develop a method for quantitative assay of alpha-Gal on pig cell surface.After centrifugation of the anticoagulated blood samples, the white blood cell layer was collected for addition of FITC-BSIB4 and analysis using FCM as well as Laser Scan Confocal Microscope (Bio-Rad).With human blood group B as negative control, and mouse myeloma SP2/0 cells as positive control. the peripheral blood cells of two Chinese pigs were all positive, there were about 9.16 x 10(5) alpha-Galactosyl epitopes expressed on the cell-membrane of granulocytes 1.37 x 10(5) on the monocytes of Banna Minipig Inbred Line (BMI), and 1.16 x 10(6) on the granulocyte and 2.45 x 10(5) on the monocytes of Sichuan White Pig (SWP). The levels of alpha-Galactosyl expression on the surface of blood cells in BMI and SWP differed in various cells, the sequence from high to lower expression levels being on the platelets, granulocytes, monocytes, erthrocytes in each species of pig.The method is useful for quantitative assay of alpha-Gal on pig cell surface, and the assay may be used for alpha-Gal for on-site large-scaled screening of living pigs.

This study was undertaken to determine whether YM injection would attenuate warm I/R injury in mice.Renal warm I/R was induced in C57BL/6 males, and the mice were sacrificed 24 hours post-reperfusion. YM was given intraperitoneally 12 hours and 30 min before I/R. The effects of YM on oxidative stress and proinflammatory mediators caused by renal I/R injury were assayed.Renal I/R caused severe injuries in the kidneys of mice. In the presence of YM at doses of 5, 15 and 25 mg/kg, the kidney malondialdehyde (MDA) content reduced by 16.6%, 25.0% and 35.5% respectively, and the kidney superoxide dismutase (SOD) activities were elevated by about 38.7%, 48.3% and 76.1% respectively. ICAM-1 expression was dramatically suppressed in the presence of YM (25 mg/kg).YM attenuates the renal warm I/R injury at least partially via decreasing the neutrophils infiltration and suppressing the over-expression of adhesion molecules.