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To observe the effect of electroacupuncture (EA) on Leptin and Leptin receptor (OB-Rb) of insulin resistant obese (OIR) rats, so as to explore its possible mechanism for obesity.Twenty-four Wistar rats were randomly divided into normal, model and EA groups, with 8 rats in each group. The OIR model was established by feeding the rats with high-fat diet for 8 weeks. EA (2 Hz, 1 mA) was applied to "Zhongwan" (CV12), "Guanyuan" (CV4), "Zusanli" (ST36) and "Fenglong" (ST40) for 30 min, once every other day for 8 weeks. At the 6th week of intervention, glucose contents of intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were measured. After 8 weeks' intervention, the body weight and food intake were recorded. Serum total cholesterol (TC) and total triglyceride (TG) were assayed by using an automatic biochemical analyzer. The expression levels of Leptin and OB-Rb proteins in the small intestine and hypothalamus were detected by Western blot.Following modeling, the body weight, food intake, IPGTT, IPITT, the contents of serum TC and TG, and the expressions of Leptin protein in small intestine and hypothalamus were significantly up-regulated (EA can improve insulin resis-tance by up-regulating the OB-Rb protein expression and enhancing the binding force of Leptin and OB-Rb.

Recent evidence indicates that long noncoding RNA colon cancer-associated transcript-1 (lncRNA CCAT1) is abundantly expressed in esophageal cancer and is closely related to the occurrence, development, invasion, metastasis, and drug resistance of this disease. However, the role and molecular mechanisms of CCAT1 in the cell proliferation and chemoresistance of esophageal cancer are largely unknown. The correlation between CCAT1 expression and drug resistance to cisplatin (CDDP) in esophageal squamous cell carcinoma (ESCC) cells was analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and quantitative real-time polymerase chain reaction (qRT-PCR) assays. CCAT1 knockdown and miR-143 overexpression or inhibition were used to verify the effects on proliferation and drug resistance via MTT, western blotting, flow cytometry, and immunofluorescence assays. qRT-PCR and western blotting were applied to detect the potential regulatory relationship among CCAT1, miR-143, PLK1, and BUBR1. A xenograft tumor assay was performed to validate the role of CCAT1 in vivo. The expression of CCAT1 was positively correlated with drug resistance in several ESCC cell lines. CCAT1 knockdown and miR-143 overexpression inhibited cell proliferation and CDDP drug resistance. Moreover, the downstream target of CCAT1 was found to be miR-143, which can regulate the expression of PLK1 and BUBR1. In vivo assays showed that CCAT1 knockdown suppressed tumor growth and enhanced the sensitivity of tumors to CDDP in nude mice. Taken together, we discovered a novel mechanism by which CCAT1 promotes cell proliferation and enhances drug resistance by regulating the miR-143/PLK1/BUBR1 signaling axis both in vitro and in vivo. Our findings further suggest that lncRNA CCAT1 may be a potential therapeutic target for overcoming chemoresistance in esophageal cancer.

Activation of NF-κB pathway and co-stimulatory system CD40/CD40L promotes the inflammation, which plays a key role in the failure of islet graft. Therefore, the purpose of this study was to determine if simultaneous blockade of CD40/CD40L and IκB/NF-κB pathways could protect islet graft. Streptozocin-induced diabetic Wistar rats were transplanted intraportally with 2000 IEQ islets isolated from Sprague-Dawley rats. The rats were divided into five groups: nontreatment group, AdGFP-treated group, Ad-IκBα-treated group, Ad-sCD40LIg-treated group, and Ad-IκBα-IRES(2) -sCD40L-treated group. The islet graft mean survival time (MST), insulin expression of islet grafts, and the levels of cytokines in peripheral blood, were measured for the animals in each group. Our study confirmed that islet cells transfected with low doses of adenovirus could achieve high transfection efficiency, and would not affect the function of islet cells (P0.05). Splenocytes cultured with Ad-IκBα-IRES2-CD40L-transfected islets resulted in homospecific hyporesponsiveness. The islet graft MST (100 d) in the Ad-IκBα-IRES2-sCD40L-treated group was dramatically prolonged compared with that in the nontreatment group (7.1 ± 1.16 d). In addition, TNF-α, IL-1β, and IFN-γ were diminished in the Ad-IκBα-IRES2-sCD40L-treated group, which was commensurate with the reduced cellular infiltration (P0.01). Simultaneous blockade of the CD40/CD40L and IκB/NF-κB pathways could effectively extend the survival of islet grafts.

To establish a more convenient and effective method for isolating adult Sertoli cells and apply the method to islet transplantation.Trypsin, DNase, collagenase and hyaluronidase were used for a one-step digestion in group A. A two-step digestion was used in group B, in which testis tissues were first digested by trypsin and DNase and then were digested by collagenase and hyaluronidase. In group C, trypsin and DNase were used in the first step of digestion, hyaluronidase was used in the second step and collagenase was used in the third step. Sertoli cells were identified by morphology and immunohistochemistry and the viability and purity of Sertoli cells were detected by MTT and flow cytometry (FCM). Expression of Fas-L was detected by Western blot and the effects of the co-transplantation of islets and Sertoli cells were compared between the three groups.Typical Sertoli cells were seen after isolation using the three different methods. Sertoli cells isolated by method A and method B were large in number while those isolated by method B and method C were pure. The results of MTT showed that the viability of Sertoli cells in group B was significantly higher than that in group A and group C (P0.05) and Western blot results showed that expression of Fas-L on Sertoli cells in group B was significantly stronger than that in group A and group C (P0.05). The purity of Sertoli cells detected by FCM in group B and group C were significantly higher than that in group A (P0.05). The survival of islets co-transplanted with Sertoli cells to renal capsule of diabetic mice was significantly longer in group B compared with that in control group as well as in group A and group C (P0.05).Sertoli cells obtained by the two-step digestion method are of higher purity and viability, which significantly prolong the survival of islet graft by co-transplantation.

To investigate the feasibility and benefits of co-culture of cryopreserved islets with small intestinal submucosa (SIS).Purified rat islets cryopreserved for one month were divided into SIS group and control group, and after culture in standard islet culture media RPMI1640 for 1 week, the morphology and function of the islets were assessed.The SIS protects the fragile islets from damage by cryopreservation, and increased the recovery from (60.6-/+3.3)% to (91.7-/+1.8) % (P0.05). Compared with the control group, incubation of the islets of the SIS group in high-glucose (16.7 mmol/L) solution resulted in significantly enhanced insulin secretion (23.7-/+1.6 vs 12.5-/+1.1 mU/L, P0.05). When the islets were incubated in high-glucose solution containing theophylline, the calculated stimulation index of SIS group was about 3-fold higher than that of the control group.Co-culture of cryopreserved rat islets with SIS can increase the recovery of islet cells and improve their function.

To investigate the role of alginate-polylysine-alginate (APA) microcapsules in protecting rat islet cells in cryopreservation.Purified rat islet cells microencapsulated with APA and free islet cells were cryopreserved for one month and then thawed for culture in RPMI 1640 overnight. The morphology of the cells was observed and their function assessed by stimulated insulin release test.APA microcapsulation protected the fragile islets from freezing damage by increasing the recovery rate of the cells from 68.6%+/-2.9% to 94.7%+/-1.4% (P0.05). After incubation with high glucose (16.7 mmol/L) solution, the insulin release from the encapsulated cells after cryopreservation significantly increased in comparison with that of the nonencapsulated cells (22.6+/-1.8 mU/L vs 11.7+/-1.5 mU/L, P0.05). In high glucose solution containing theophylline, the calculated stimulation index of the encapsulated cells was about 3 times that of the nonencapsulated cells.APA microencapsulation may significantly increase the post-thaw recovery and improve the function for cryopreserved rat islets.

To evaluate the effect of HSGJ on chronic allograft nephropathy (CAN) using standard rat model of CAN.Renal transplantation was performed with Fisher rats as donors and Lewis rats as recipients. All the recipients were randomly divided into control group and medication groups (high and low dosage of HSGJ, fed every other day). After 16 weeks of treatment, renal function and the histological alteration of CAN were measured. The expression of the TGFbeta1 mRNA in the allograft was evaluated by real-time PCR.The content of 24 h urine protein and the level of serum creatinine in the medication groups were significantly decreased (P0.01) as compared with control group, whereas the creatinine clearance was increased (P0.01). The degree of glomerular sclerosis and the Banff score of medication groups were lower than the control group respectively (P0.01), in consistent with decreased expression of the TGF 1mRNA.HSGJ can prevent the chronic allograft nephropathy and the mechanism may be related with its influence on the expression of the TGFbeta1.

The ability to maintain isolated human islet preparation in tissue culture has recently been adopted by most islet transplant centers to improve the safety and practicality of islet transplantation. However, maintaining islet viability and recovery remains a challenge in clinical setting. Extracellular matrix (ECM) is one of the most important components of islet microenvironment. The reconstruction of the cell-matrix relationship seems to be effective in improving the loss of differentiated islet structure and function. Small intestinal submucosa (SIS), a naturally occurring ECM, has been investigated to be able to promote wound healing, tissue remodeling, and cell growth. The purpose of this study was to evaluate the recovery and function of isolated rat pancreatic islets after in vitro culture with SIS.Pancreatic islets were isolated from Wistar rats by using standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation, and EuroFicoll purification. Groups of purified islets were cultured in plates which were coated with multilayer SIS (SIS-treated group) or without (standard cultured group) for 7 days and 14 days in standard islet culture conditions of RPMI 1640 tissue culture media in humidified atmosphere containing 95% air and 5% CO2 at 37 degree centigrade. The mean recovery of islets after the culture period was determined by sizing duplicate counts of a known volume and their viability was assessed by static incubation with low glucose (2.7 mmol), high glucose (16.7 mmol) and high glucose solution supplemented with 50 mum 3-isobutyl-1-methylxanthine (IBMX) solution.After 7 days and 14 days of in vitro tissue culture, the SIS-treated group showed a significantly higher recovery compared with those cultured under standard conditions. The recovery in the SIS-treated group was about two times of the control group cultured in standard conditions after 14 days culture. In the SIS-treated group, there was no statistically difference between the short and long periods of culture(95.8+/-1.0% vs. 90.8+/-1.5%, P0.05). During incubation in high glucose (16.7 mmol) solution, there was a 2-3 fold increase in insulin secretion from both groups,but the SIS-treated group showed a higher increase than the standard cultured group after 14-day culture (20.7+/-1.1 mU/L vs. 11.8+/-1.1 mU/L, P0.05). When islets were placed in the high glucose solution supplemented with IBMX, the stimulated insulin response in the SIS-treated group was higher than that in the standard cultured group in spite of the duration of the culture. The stimulation index of the SIS-treated group was about 2-3 times of the standard cultured group. In addition, after a long period of culture, the stimulation index of the SIS-treated group was statistically equivalent with that of the short period of culture (9.5+/-0.2 vs. 10.2+/-1.2, P0.05).The co-culture of isolated rat islets with native sheet-like SIS can provide an excellent extracellular matrix, possible biotrophic and growth factors that promote the recovery and subsequent function of islets after in vitro tissue culture. In view of results of this study and rapid degradation of SIS in vitro, future studies will investigate the extended duration of culture and the effect of SIS on islets in vitro.