Search

Copyright of Journal of Zhejiang University: Science B is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4+ CD25+ regulatory T (Treg) cells, in order to inform further studies in Treg cell function. We separately used magnetic cell sorting and flow cytometry sorting to identify CD4+ CD25+ Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4+ CD25+ Treg cells, trypan blue staining to detect cell viability, and propidium iodide (PI) staining to assess the cell viability. We detected the immune inhibition of CD4+ CD25+ Treg cells in the in vitro proliferation experiments. The results showed that compared to flow cytometry sorting, magnetic cell sorting took more time and effort, but fewer live cells were obtained than with flow cytometry sorting. The CD4+ CD25+ Treg cells, however, obtained with both methods have similar immunosuppressive capacities. The result suggests that both methods can be used in isolating CD4+ CD25+ Treg cells, and one can select the best method according to specific needs and availability of the methodologies. [ABSTRACT FROM AUTHOR]

Objective: to study the relationship between the expression of serum human leucocyte antigen-G5 (HLA-G5)/soluble CD30 (sCD30) and the function of renal graft in kidney transplant recipients and investigate the immune status of recipients with combined HLA-G5 and sCD30., Methods: from January 2002 to November 2008, a total of 66 kidney transplant recipients in our centre were selected as subjects and divided into three groups: stable function of renal graft (n = 38), acute rejection (n = 15) and chronic rejection (n = 13). The expressions of serum HLA-G5 and sCD30 were detected. There were two different immune conditions with acute/chronic allograft rejection and normal renal graft in kidney transplant recipients as evaluated by combined HLA-G5 and sCD30. The sensitivity, specificity and critical value of the method were analyzed by the curve of receiver operating characteristic., Results: the levels of HLA-G5 and sCD30 were significantly correlated with serum creatinine (r = -0.493, 0.691, both P < 0.01). Within the first year post-transplantation, the sensitivity was 78.6% and the specificity 85.7% when HLA-G5 critical value 82 microg/L and sCD30 critical value 12.2 microg/L. After one year post-transplantation: the sensitivity was 92.3% and the specificity 84.6% when HLA-G5 critical value 141 microg/L and sCD30 critical value 10.3 microg/L., Conclusion: the immune state of recipients are evaluated by combine HLA-G5 and sCD30 which may be a simple and valid method.

Objective: To construct a replication-incompetent recombinant adenovirus mediating short hairpin RNA (shRNA)-induced tissue factor gene silencing in the islet., Methods: Four pairs of complementary oligonucleotides were designed and synthesized to create double-stranded oligonucleotides (ds oligo). The ds oligos were cloned into Pentr/U6 vector to construct the shuttle plasmid pENTR/U6-shRNA, which was transduced into human islets via liposome after sequence verification. The plasmid with the best silencing effect was identified by real-time RT-PCR, followed by homologous recombination with the adenovirus backbone plasmid. The functional clone was transfected into 293A cells to amplify the adenovirus, whose silencing effect against TF expression was tested using real-time RT-PCR and Western blotting., Results: The pENTR/U6-shRNA shuttle plasmid was constructed and verified by sequencing. The recombinant adenovirus-mediated shRNA against TF was constructed, and real-time RT-PCR and Western blotting demonstrated that the strongest silencing effect of the adenovirus against TF occurred on the 4th day following islet transfection., Conclusion: Replication-incompetent recombinant adenovirus-mediated shRNA against TF has been successfully constructed, which has good silencing effect against TF expression in human islet in vitro.

Objective: To establish a stable method for obtaining large quantity of highly purified immature dendritic cells (imDCs) in vitro, and identify the morphology, function and surface markers of the cells., Methods: CD117(+) hemopoietic stem cells (HSCs) were isolated and purified from the bone marrow of healthy C57 mice by magnetic affinity cell sorting. After cell expansion by treatment with stem cell factor (SCF) and interleukin-3 (IL-3), the HSCs were induced for directional differentiation into imDCs by treatment with GM-CSF, IL-4 and IL-10. The imDCs obtained were identified by morphological and functional observation under inverted microscope, scanning electron microscope and transmission electron microscope, followed by detection of the expressions of the surface markers using flow cytometry., Results: After 3, 5 and 7 days of culture in the presence of SCF+IL-3, the cells were expanded by 10.34-/+1.43, 22.65-/+2.71 and 54.39-/+3.08 folds, respectively. The HSCs were successfully induced to differentiate into imDCs with phagocytotic activity. The dendrites of the imDCs were short small, and appearing spinous. The expressions of surface markers were detected from the cells showing the phenotype of CD11c(+), I-A/I-E(low), CD40(-), CD80(-), CD86(-)., Conclusion: The method described allows steadily acquisition of large quanty of highly purified imDCs and of their effective identification in vitro.

Background: In the mouse skin allograft model, specific immune tolerance to the donor was induced by injection of donor hepatic non-parenchymal cells (NPCs). This markedly prolonged the survival time of the allograft. The mechanism of the induction of immune tolerance with donor hepatic NPCs is thought to be related to microchimerism and the IL-4 level. This work aimed at exploring the way of inducing immune tolerance and understanding the mechanism., Methods: C57BL/6 (B6) mice were primed by intravenous injection of 2 x 10(7) NPCs from C3H mice. Cyclophosphamide (200 mg/kg) was injected intraperitoneally 48 hours later. Eighteen days after the NPC injection, skin from C3H mice was transplanted to B6 mice and the survival of the grafts was assessed. The immune reaction of splenocytes from the treated B6 mice to donor-specific T-cells was measured by 3H-TdR incorporation. Microchimerism in the spleen was determined by flow cytometric analysis sytem (FCAS) analysis, and the serum level of IL-4 was assayed by ELISA at designed times., Results: The survival time of the skin graft was markedly prolonged from 10 days to 70 days in controls. Microchimerism in the spleen was found as early as day 1 post-NPC injection, then it increased steadily, and there was a positive relationship between graft survival and the quantity of microchimerism. The ELISA results showed that NPC infusion enhanced IL-4 production, especially in the mice with longer graft survival., Conclusion: Donor NPC infusion pre-transplant can prolong the survival of the skin graft and microchimerism and high levels of IL-4 may be involved.

Objective: To study the protective effect of recombinant adenovirus-mediated human cytosolic glutathione peroxidase (hCGPx) gene transfection on vascular endothelial cells ECV304 from oxidative damage., Methods: pGEM-T Easy Vector containing hCGPx cDNA and recombinant adenovirus shuttle plasmid pACCMV-pLpA were used to construct the shuttle plasmid pACCMV-hCGPx for cotransfection of 293 cells with pJM17, thereby to obtain the recombinant adenovirus AdCMV-hCGPx. Cultured ECV304 cells were transfected with AdCMV-hCGPx for 24, 48 and 72 h, respectively, with the cells transfected with the empty vector serving as control, and hCGPx gene expression was then examined in the transfected cells. The transfected cell viability and apoptotic cell ratio were evaluated after treatment of the cells with H(2)O(2)., Results: The expression ratio of hCGPx gene was significantly higher in the AdCMV-hCGPx-transfected cells than in those with empty vector transfection (P<0.01). The hCGPx gene-transfected cells showed significantly higher viability and significantly lower apoptotic ratio than the control cells following challenge with H(2)O(2)-induced oxidative damage., Conclusion: hCGPx gene transfer mediated by recombinant adenovirus protects the vascular endothelial cells from oxidative damage in vitro, possibly due to the antioxidative and apoptosis-inhibiting effect of hCGPx.

Aim: To evaluate the recovery and function of isolated rat pancreatic islets during in vitro culture with small intestinal submucosa (SIS)., Methods: Pancreatic islets were isolated from Wistar rats by standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation and Euroficoll purification. Purified islets were cultured in plates coated with multilayer SIS (SIS-treated group) or without multilayer SIS (standard cultured group) for 7 and 14 d in standard islet culture media of RPMI 1640. After isolation and culture, islets from both experimental groups were stained with dithizone and counted. Recovery of islets was determined by the ratio of counts after the culture to the yield of islets immediately following islet isolation. Viability of islets after the culture was assessed by the glucose challenge test with low (2.7 mmol/L) and high glucose (16.7 mmol/L) solution supplemented with 50 mmol/L 3-isobutyl-1-methylxanthine (IBMX) solution. Apoptosis of islet cells after the culture was measured by relative quantification of histone-complexed DNA fragments using ELISA., Results: After 7 or 14 d of in vitro tissue culture, the recovery of islets in SIS-treated group was significantly higher than that cultured in plates without SIS coating. The recovery of islets in SIS-treated group was about twice more than that of in the control group. In SIS-treated group, there was no significant difference in the recovery of islets between short- and long-term periods of culture (95.8+/-1.0% vs 90.8+/-1.5%, P>0.05). When incubated with high glucose (16.7 mmol/L) solution, insulin secretion in SIS-treated group showed a higher increase than that in control group after 14 d of culture (20.7+/-1.1 mU/L vs 11.8+/-1.1 mU/L, P<0.05). When islets were placed in high glucose solution containing IBMX, stimulated insulin secretion was higher in SIS-treated group than in control group. Calculated stimulation index of SIS-treated group was about 23 times of control group. In addition, the stimulation index of SIS-treated group remained constant regardless of short- and long-term periods of culture (9.5+/-0.2 vs 10.2+/-1.2, P>0.05). Much less apoptosis of islet cells occurred in SIS-treated group than in control group after the culture., Conclusion: Co-culture of isolated rat islets with native sheet-like SIS might build an extracellular matrix for islets and provide possible biotrophic and growth factors that promote the recovery and subsequent function of islets.

Background: Globally, 180 million people suffer from diabetes mellitus. Islet transplantation is believed to be an almost ideal therapy for insulin-dependent patients. How to maintain the viability and the function of isolated human islets is a challenge in clinical practice. Sertoli cells are considered 'nurse cells' in the seminiferous tubules and have been used in cell graft protocols for neurodegenerative diseases and diabetes in many studies. Many researchers have used immature murine testes as the primarily source of Sertoli cells in islet transplantation because they are easily purified. Mature human Sertoli cells have been seldom investigated. In the present study, we developed a method for the isolation and culture of Sertoli cells derived from adult human testes, and investigated their effects on the function of allogeneic islets when they were cultured together in vitro., Methods: Adult Sertoli cells were prepared successfully by two-step enzyme digestion with trypsin, collagenase and hyaluronidase. They were identified by morphological characteristics and their activity was determined by MTT colorimetry over a 28-day culture time in vitro. A glucose-stimulated insulin secretion test was performed to detect the effects of Sertoli cells on allogeneic islets' function when they were co-cultured for 21 days in vitro., Results: In cultured cells, mature human Sertoli cells accounted for more than 90% of total cells. The activity of Sertoli cells reached 95% and they remained highly cytoactive for a long time in vitro (P > 0.05). Compared with the islets cultured alone, the co-cultured islets with allogeneic Sertoli cells maintained higher sensitivity to glucose stimulation for the duration of the experiment (P < 0.01)., Conclusions: A method of isolation and culture of Sertoli cells from adult testes has been established. Sertoli cells could enhance allogeneic islets' function when they were co-cultured in vitro. They could be a helper cell in islet transplantation.

Background: 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., Methods: 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., Results: 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%, P>0.05). 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, P<0.05). 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, P>0.05)., Conclusions: 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.