Immunoprotection Of Islet Transplantation With Neonatal Porcine Sertoli Cells And Its Immuneprivilege Mechanism

Partâ… Neonatal Porcine Sertoli cells Survival in Non-immunosuppressiveWistar Rats【Objective】Evaluate neonatal porcine Sertoli cells (NPSCs) survival in thenon-immunosuppressive Wsitar rats and investigate its mechanism.【Methods】Neonatal porcine Sertoli cells were isolated from 10 to 15 daysold male largewhite pigs.The isolated NPSCs were cultured for 3 days before testing for Sox9 and FasLby RT-PCR (reverse transcription polymerase chain reaction,RT-PCR) and using fortransplantation.1.5×10⁶ of NPSCs alone were implanted under the left renal capsule of each Wistar rat in the absence of immunosuppression.The xenograft-bearing kidneys wereharvested at 3,7,14,21,and 40 days post-transplant and then examined the expression ofSox9 with the immunochemistry and RT-PCR.【Results】With Sox9 immunostaining,about 5.68±1.75×10⁷ NPSCs were obtained fromeach testes which represented more than 90% of the total isolated cells.Interestingly,FasLwhich was thought to confer Sertoli cells immunological privilege was almost negative.After transplantation,both immunochemistry and RT-PCR indicated that Sox9 in graftscould be detected at 3,7,14,and 21 days post-transplant,however,it was nearly negative at40 days post-transplant.【Conclusion】Neonatal porcine Sertoli cells could survive more than 21 days but less than40 days in non-immunosuppressive Wsitar rats.FasL did not contribute to theimmunoprotection provided by NPSCs.Partâ…¡Isolation Islets of SD Rats and Induction Chemically DiabeticModel in Wistar Rats【Objective】To isolate islets of SD rats with optimized Shapiro methods and inducechemically diabetes in Wistar rats.【Methods】Islets were isolated from SD rats with 1 mg/ml collagenase type V contained7.5mmol/L Ca²⁺ digestion,and then purified by Ficoll 400 discontinuous density gradientcentrifugation.DTZ staining was used to identify the isolated islets.Wistar rats wererendered diabetic with alloxan at a dose of 200mg/kg by intraperitoneal injection.Onlythose animals exhibiting two consective non-fasting blood glucose values≥22mmol/Lwere considered as diabetes.【Results】About 520±30 IEQ islets were obtained from each rats by our optimizedmethods.With DTZ staining identification,the islets were represented more than 80% of the isolated cells.After hand-picked,the purity of islets would be more than 90%.Almost85% Wistar rats with alloxan adimistration were induced into diabetes mellitus.【Conclusion】An enrich fraction islets were efficiallyand conveniently obtained with ouroptimized digestion methods.A single intraperitoneal injection of alloxan could stablyinduce chemically diabetes in Wistar rats.Partâ…¢Cotransplantation with Xeno-neonatal Porcine Sertoli cellsSignificantly Prolongs Islet AIIograft Survival inNon-immunosuppressive Rats【Objective】To investigate neonatl porcine Sertoli cells protect islet allograft survival innon-immunosuppressive rats and its mechanisms.【Methods】Diabetic Wistar recipient rats were divided into follow groups.Group 1 (n=8),1500 IEQ SD islets alone was implantated under the left renal capsule of recipient.Group 2(n=8),1500 IEQ SD islets in combination with 1.5×10⁶NPSCs were implanted under theleft renal capsule of recipient.Group 3 (n=8),1500 IEQ SD islets with 1.0×10⁷NPSCs wereco-transplanted under the left renal capsule of recipient.Group 4 (n=5),1500 IEQ SD isletswere implanted under the left renal capsule and 1×107 of NPSCs were transplanted underthe right renal capsule of the same recipient rat.Blood samples were obtained from the tailvein of non-fasted animals for glucose assay.Graft-bearing kidneys (n=3) were harvested atthe time of rejection (return to hyperglycemia,two consecutive readings of≥11.2mmol/Lor at 7 days post transplant,and then analysis the pathology and expression of Sox9,insulin,Bcl-2 and HO-1.【Results】Although in combination with 1.5×10⁶NPSCs,mean graft survival times were8.33±0.58 days,while co-transplanted with 1.0×10⁷NPSCs,mean graft survival timessignificantly increased to 16.33±1.53 days (P＜0.05 vs.islet alone,5.67±0.94 days). However,when 1,500 IEQ SD islets and 1.0×10⁷NPSCs were transplanted seperately tothe different site of the same recipient,mean survival time,was only 5.25±0.5 days( P＞0.05 vs.islets alone).With the immunopathology analysis,augmented islet survival wasassociated with reduced lymphocytic infiltrate as well as elevated numbers of sox9 positivecells and induced expression of Bcl-2 in transplanted sites.【Conclusion】Enough neonatl porcine Sertoli cells prolonged islet allograft survival innon-immunosuppressive rats with induced expression of Bcl-2 in local.Partâ…£Resistance of Neonatal Porcine Sertoli cells to Xeno-antibodiesMediated Complement Lysis【Objective】To invesitigate whether neonatal porcine Sertoli cells could protectthemselves from humoral injury mediated by xenoreactive antibodies and complement.【Methods】The SV40-PED was served as control cells.α-Gal expression of NPSCs wasmeasured FACS and immunofluorescence with FITC-GS-IB4 staing.The binding of humanserum IgG and IgM with NPSCs was assayed by FACS.After the incubation of NPSCswith 20% human B serum in vitro,the cellular lysis and viability assay were detected withLDH assay and MTT method,and then activation of the complement cascade (C3,C4,andC5b-9) was examined by immunohistochemistry and immunofluorescence.Western blotwere used to examine the expression of clusterin on the two cells,then CD59 and CD46were detected by RT-PCR.【Results】α-Gal expression was found on NPSCs,however,its mean fluorescenceintensity (MFI) was only 41.78±2.39 (P＜0.01 vs.SV40-PED,95.17±2.39).Both of the twocells could bind with the IgG,IgM,however,MFI of IgM and IgG bind with NPSCs wassignificantly lower than SV40-PED.After incubation with 20% human serum,cellular lysisratio of the NPSCs was significantly lower than SV40-PED (24.38%±0.50% vs 53.13%±14.53%,P＜0.01) and the viability of NPSCs and SV40-PED were98.73%±18.84% and 52.43%±8.08%,P＜0.01,respectively.With immunohistochemistryand immunofluorescence analysis,C3 and C4 were found binding on both the NPSCs andSV40-PED cells,however,C5b-9 was only detected on SV40-PED cells.Additionally'compared with SV40-PED,expression of clusterin and CD59 on NPSCs tends to increasedby western blot or RT-PCR,however,expression of CD46 has no difference.【Conclusion】NPSCs could survive and resist xenoreactive antibodies mediatedcomplement lysis in vitro by prevent the complement attack complex formation.This mayassociate with NPSCs expressed low level ofα-Gal,and then lead to lower binding of thexenoreactive antibodies,and also expressed higher level of clusterin and CD59.