新生猪Sertoli细胞在胰岛移植中的免疫保护作用及其免疫豁免机制研究
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摘要
第一部分新生猪Sertoli细胞在Wistar大鼠肾包膜下存活的实验研究
【目的】在不使用任何免疫抑制剂和不进行任何免疫保护干预的措施下，探讨新生猪Sertoli细胞(NPSCs)在Wistar大鼠肾包膜下的生存状态并初步研究其自身免疫豁免机制，为NPSCs与胰岛细胞共同移植的应用奠定理论基础。
【方法】体外分离培养10～15日龄的湖北白猪睾丸Sertoli细胞即NPSCs；该细胞体外培养3天后，应用RT-PCR检测Sox9和FasL的表达；同时将1．5×10~6个NPSCs移植入正常Wistar大鼠左肾包膜下，术后分别在第3、7、14、21和40天切取Wistar大鼠左侧肾脏，采用RT-PCR和免疫组化染色检测肾包膜下移植的NPSCs特异性标记Sox9的表达。
【结果】每个新生猪睾丸可以获得5．68±1．75×10~7个NPSCs，占培养细胞总数的90％左右，活性达95％；体外分离培养的NPSCs能够稳定表达其特异性标记因子Sox9，但FasL几乎不表达。1．5×10~6个NPSCs移植入Wistar大鼠左肾包膜下第3、7、14和21天后，RT-PCR证实肾包膜下的移植物内有大量猪Sox9表达，Sox9免疫组化染色亦表明肾包膜下有成团聚集的Sox9阳性细胞；但是移植后第40天时，RT-PCR证实肾包膜下移植物内的猪Sox9表达明显减弱，同时免疫组化表明成团聚集的Sox9阳性细胞团明显减少，NPSCs仅散在存在。
【结论】在不使用任何免疫抑制剂和不进行任何免疫保护干预的措施下，NPSCs在Wistar大鼠肾包膜下可以存活21天以上，但移植后40天时存活的NPSCs数量明显减少，其免疫豁免机制与FasL的作用无关。
第二部分SD大鼠胰岛分离纯化及Wistar大鼠化学性糖尿病模型的建立
【目的】优化Shapiro的胰岛细胞分离方法，分离纯化Sprague Dawley(SD)大鼠胰岛细胞；建立Wistar大鼠化学性糖尿病模型，为胰岛细胞移植研究奠定技术支持。
【方法】采用含7．5mmol／LCa~(2+)的1mg／L的V型胶原酶胰管逆行灌注胰腺、37℃静止消化，Ficoll 400不连续密度梯度离心纯化的方法，分离培养SD大鼠胰岛细胞；培养的胰岛细胞经双硫腙(dithizon，DTZ)染色鉴定，ELISA葡萄糖刺激实验检测分离培养的胰岛细胞功能；200mg／kg的四氧嘧啶一次性腹腔注射诱导Wistar大鼠化学性糖尿病模型，连续两次非空腹血糖≥22mmol／L视为造模成功。
【结果】每只大鼠可获得520±30个胰岛当量(islets equivalent quantity，IEQ)的胰岛细胞，DTZ染色鉴定后见胰岛细胞纯度达80％，手检后纯度达90％以上；Wistar大鼠腹腔注射四氧嘧啶后，第3和7天非空腹血糖均大于22mmol／L，并且出现多饮、多尿、体重下降等糖尿病症状，造模成功率达85％。
【结论】经优化后的分离方法更加简捷并获得了纯度较高、大量的大鼠胰岛细胞；通过单次腹腔注射四氧嘧啶能够稳定、高效的诱导大鼠化学性糖尿病模型。
第三部分联合移植异种新生猪Sertoli细胞延长大鼠同种异体胰岛移植存活
【目的】探讨异种NPSCs在大鼠同种异体胰岛细胞移植中的免疫保护作用及潜在的免疫豁免机制，为NPSCs在临床同种异体胰岛移植的应用奠定理论基础。
【方法】在上述第一、二部分的实验基础上，建立SD大鼠→Wistar糖尿病受鼠的同种异体胰岛移植模型。实验分组如下：①单纯胰岛细胞移植组(n=8)，1500 IEQ的SD大鼠胰岛细胞移植入Wistar糖尿病大鼠左肾包膜下；②小量NPSCs与胰岛细胞同侧移植组(n=8)，1．5×10~6个NPSCs与1500 IEQ的SD大鼠胰岛细胞共同移植入糖尿病Wistar大鼠左肾包膜下；③大量NPSCs与胰岛细胞同侧移植组(n=8)，1．0×10~7个NPSCs与1500 IEQ的SD大鼠胰岛细胞共同移植入糖尿病Wistar大鼠左肾包膜下；④大量NPSCs与胰岛细胞分侧移植组(n=5)，1．0×10~7个NPSCs移植入Wistar糖尿病大鼠右肾包膜下，同时将1500 IEQ的SD大鼠胰岛细胞移植入另一侧左肾包膜下；术后观察Wistar糖尿病大鼠血糖变化，血糖连续2次≥11．2mmol／L时视为胰岛细胞发生排斥；术后7天或移植物发生排斥时，免疫病理学比较肾包膜下淋巴细胞浸润情况并检测胰岛细胞特异性标记物Insulin、NPSCs特异性标记因子Sox9和保护性基因Bcl-2、HO-1的表达。
【结果】1．5×10~6个NPSCs与1500 IEQ胰岛细胞共同移植入受鼠左肾包膜后，胰岛移植物平均存活时间仅稍微延长到8．33±0．58天；当同侧共同移植的NPSCs增加到1．0×10~7个时，胰岛移植物平均存活时间显著延长到16．33±1．53天，与单纯胰岛移植组(5．67±0．94天)相比，差异有显著意义(P＜0．01)；但是将1．0×10~7个NPSCs移植入受体鼠右肾，同时将1500 IEQ胰岛细胞移植入另一侧即左肾后，胰岛移植物平均存活时间未见明显延长(5．25±0．25天)，与单纯胰岛移植组相比差异无显著意义(P＞0．05)。大量NPSCs与胰岛细胞同侧移植组在移植术后7天，与单纯胰岛移植组相比，免疫组化可见，肾包膜炎症反应轻微，淋巴细胞散在浸润且局限在肾包膜下，包膜内可见呈团块状排列的细胞团，经Sox9免疫组化染色证实该细胞团即为移植的NPSCs；Insulin染色可见肾包膜下有大量的胰岛细胞存在；此外免疫组化还证实，大量NPSCs组肾包膜下有大量Bcl-2保护性基因的表达，但HO-1在各组间均有表达。
【结论】足量NPSCs对共同移植的大鼠胰岛细胞具有局部的免疫学保护作用，并显著延长大鼠胰岛细胞在同种异体肾包膜下的存活时间；其机制可能与NPSCs在局部诱导保护性基因Bcl-2表达上调有关，而与FasL的作用无关。NPSCs和胰岛细胞不同部位移植则无保护作用。
第四部分新生猪Sertoli细胞抵御人血清中天然抗体介导的补体杀伤作用
【目的】探讨异种NPSCs抵御人血清中天然抗体介导的补体杀伤作用及其主要机制，为NPSCs与胰岛细胞联合移植的临床应用奠定理论基础。
【方法】以永生化猪血管内皮细胞系(SV40-PED)作为对照，FITC-GS-IB4 lectin染色流式细胞仪(FACS)及免疫荧光检测并比较NPSCs与SV40-PED天然抗原α-Gal的表达；FACS检测并比较2种细胞分别与正常人血清(normal human serum，NHS)中天然抗体IgM、IgG的结合情况；乳酸脱氢酶释放试验(LDH法)检测20％NHS对NPSCs及SV40-PED细胞的杀伤；MTT法检测两种细胞在20％NHS中培养时的活性；细胞免疫荧光检测补体C3c、C4d在NPSCs、SV40-PED的沉积；细胞免疫组化及免疫荧光检测补体攻膜复合物C5b-9在2种细胞上的沉积。Western blot检测并比较2种细胞clusterin的表达；RT-PCR检测并比较两种细胞补体调节蛋白CD46、CD59的表达。
【结果】NPSCs能够表达α-Gal，但明显弱于SV40-PED(平均荧光强度分别为41．78±2．39和95．17±2．39，P＜0．05)；NPSCs虽可与人血清中天然抗体IgG、IgM结合，但其结合程度明显弱于SV40-PED；20％NHS对NPSCs、SV40-PED的杀伤率分别为24．38％±0．50％和53．13％±14．53％(P＜0．01)；2种细胞在20％NHS中的活性分别为：98．73％±18．84％和52．43％±8．08％(P＜0．01)；免疫荧光及组化可见SV40-PED细胞上分别有补体C3c、C4d和C5b-9的沉积；但NPSCs仅有C3c和C4d沉积，而未见C5b-9沉积；Western blot证明，clusterin在NPSCs的表达略强于SV40-PED；RT-PCR证实，NPSCs表达的CD59显著强于SV40-PED，而CD46在此两种细胞的表达无明显差异。
【结论】NPSCs可以显著抵御人血清中天然抗体介导的杀伤作用，其机制可能与NPSCs低表达α-Gal导致其与天然抗体结合较少并高表达clusterin及CD59等补体调节蛋白从而抑制补体攻膜复合物形成有关。
PartⅠNeonatal 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.1.5×10~6 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±1.75×10~7 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.
PartⅡIsolation 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 Ca~(2+) 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.
PartⅢCotransplantation 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 1.5×10~6NPSCs were implanted under theleft renal capsule of recipient.Group 3 (n=8),1500 IEQ SD islets with 1.0×10~7NPSCs 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 1.5×10~6NPSCs,mean graft survival times were8.33±0.58 days,while co-transplanted with 1.0×10~7NPSCs,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 1.0×10~7NPSCs 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.
PartⅣResistance 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.
【目的】在不使用任何免疫抑制剂和不进行任何免疫保护干预的措施下，探讨新生猪Sertoli细胞(NPSCs)在Wistar大鼠肾包膜下的生存状态并初步研究其自身免疫豁免机制，为NPSCs与胰岛细胞共同移植的应用奠定理论基础。
【方法】体外分离培养10～15日龄的湖北白猪睾丸Sertoli细胞即NPSCs；该细胞体外培养3天后，应用RT-PCR检测Sox9和FasL的表达；同时将1．5×10~6个NPSCs移植入正常Wistar大鼠左肾包膜下，术后分别在第3、7、14、21和40天切取Wistar大鼠左侧肾脏，采用RT-PCR和免疫组化染色检测肾包膜下移植的NPSCs特异性标记Sox9的表达。
【结果】每个新生猪睾丸可以获得5．68±1．75×10~7个NPSCs，占培养细胞总数的90％左右，活性达95％；体外分离培养的NPSCs能够稳定表达其特异性标记因子Sox9，但FasL几乎不表达。1．5×10~6个NPSCs移植入Wistar大鼠左肾包膜下第3、7、14和21天后，RT-PCR证实肾包膜下的移植物内有大量猪Sox9表达，Sox9免疫组化染色亦表明肾包膜下有成团聚集的Sox9阳性细胞；但是移植后第40天时，RT-PCR证实肾包膜下移植物内的猪Sox9表达明显减弱，同时免疫组化表明成团聚集的Sox9阳性细胞团明显减少，NPSCs仅散在存在。
【结论】在不使用任何免疫抑制剂和不进行任何免疫保护干预的措施下，NPSCs在Wistar大鼠肾包膜下可以存活21天以上，但移植后40天时存活的NPSCs数量明显减少，其免疫豁免机制与FasL的作用无关。
第二部分SD大鼠胰岛分离纯化及Wistar大鼠化学性糖尿病模型的建立
【目的】优化Shapiro的胰岛细胞分离方法，分离纯化Sprague Dawley(SD)大鼠胰岛细胞；建立Wistar大鼠化学性糖尿病模型，为胰岛细胞移植研究奠定技术支持。
【方法】采用含7．5mmol／LCa~(2+)的1mg／L的V型胶原酶胰管逆行灌注胰腺、37℃静止消化，Ficoll 400不连续密度梯度离心纯化的方法，分离培养SD大鼠胰岛细胞；培养的胰岛细胞经双硫腙(dithizon，DTZ)染色鉴定，ELISA葡萄糖刺激实验检测分离培养的胰岛细胞功能；200mg／kg的四氧嘧啶一次性腹腔注射诱导Wistar大鼠化学性糖尿病模型，连续两次非空腹血糖≥22mmol／L视为造模成功。
【结果】每只大鼠可获得520±30个胰岛当量(islets equivalent quantity，IEQ)的胰岛细胞，DTZ染色鉴定后见胰岛细胞纯度达80％，手检后纯度达90％以上；Wistar大鼠腹腔注射四氧嘧啶后，第3和7天非空腹血糖均大于22mmol／L，并且出现多饮、多尿、体重下降等糖尿病症状，造模成功率达85％。
【结论】经优化后的分离方法更加简捷并获得了纯度较高、大量的大鼠胰岛细胞；通过单次腹腔注射四氧嘧啶能够稳定、高效的诱导大鼠化学性糖尿病模型。
第三部分联合移植异种新生猪Sertoli细胞延长大鼠同种异体胰岛移植存活
【目的】探讨异种NPSCs在大鼠同种异体胰岛细胞移植中的免疫保护作用及潜在的免疫豁免机制，为NPSCs在临床同种异体胰岛移植的应用奠定理论基础。
【方法】在上述第一、二部分的实验基础上，建立SD大鼠→Wistar糖尿病受鼠的同种异体胰岛移植模型。实验分组如下：①单纯胰岛细胞移植组(n=8)，1500 IEQ的SD大鼠胰岛细胞移植入Wistar糖尿病大鼠左肾包膜下；②小量NPSCs与胰岛细胞同侧移植组(n=8)，1．5×10~6个NPSCs与1500 IEQ的SD大鼠胰岛细胞共同移植入糖尿病Wistar大鼠左肾包膜下；③大量NPSCs与胰岛细胞同侧移植组(n=8)，1．0×10~7个NPSCs与1500 IEQ的SD大鼠胰岛细胞共同移植入糖尿病Wistar大鼠左肾包膜下；④大量NPSCs与胰岛细胞分侧移植组(n=5)，1．0×10~7个NPSCs移植入Wistar糖尿病大鼠右肾包膜下，同时将1500 IEQ的SD大鼠胰岛细胞移植入另一侧左肾包膜下；术后观察Wistar糖尿病大鼠血糖变化，血糖连续2次≥11．2mmol／L时视为胰岛细胞发生排斥；术后7天或移植物发生排斥时，免疫病理学比较肾包膜下淋巴细胞浸润情况并检测胰岛细胞特异性标记物Insulin、NPSCs特异性标记因子Sox9和保护性基因Bcl-2、HO-1的表达。
【结果】1．5×10~6个NPSCs与1500 IEQ胰岛细胞共同移植入受鼠左肾包膜后，胰岛移植物平均存活时间仅稍微延长到8．33±0．58天；当同侧共同移植的NPSCs增加到1．0×10~7个时，胰岛移植物平均存活时间显著延长到16．33±1．53天，与单纯胰岛移植组(5．67±0．94天)相比，差异有显著意义(P＜0．01)；但是将1．0×10~7个NPSCs移植入受体鼠右肾，同时将1500 IEQ胰岛细胞移植入另一侧即左肾后，胰岛移植物平均存活时间未见明显延长(5．25±0．25天)，与单纯胰岛移植组相比差异无显著意义(P＞0．05)。大量NPSCs与胰岛细胞同侧移植组在移植术后7天，与单纯胰岛移植组相比，免疫组化可见，肾包膜炎症反应轻微，淋巴细胞散在浸润且局限在肾包膜下，包膜内可见呈团块状排列的细胞团，经Sox9免疫组化染色证实该细胞团即为移植的NPSCs；Insulin染色可见肾包膜下有大量的胰岛细胞存在；此外免疫组化还证实，大量NPSCs组肾包膜下有大量Bcl-2保护性基因的表达，但HO-1在各组间均有表达。
【结论】足量NPSCs对共同移植的大鼠胰岛细胞具有局部的免疫学保护作用，并显著延长大鼠胰岛细胞在同种异体肾包膜下的存活时间；其机制可能与NPSCs在局部诱导保护性基因Bcl-2表达上调有关，而与FasL的作用无关。NPSCs和胰岛细胞不同部位移植则无保护作用。
第四部分新生猪Sertoli细胞抵御人血清中天然抗体介导的补体杀伤作用
【目的】探讨异种NPSCs抵御人血清中天然抗体介导的补体杀伤作用及其主要机制，为NPSCs与胰岛细胞联合移植的临床应用奠定理论基础。
【方法】以永生化猪血管内皮细胞系(SV40-PED)作为对照，FITC-GS-IB4 lectin染色流式细胞仪(FACS)及免疫荧光检测并比较NPSCs与SV40-PED天然抗原α-Gal的表达；FACS检测并比较2种细胞分别与正常人血清(normal human serum，NHS)中天然抗体IgM、IgG的结合情况；乳酸脱氢酶释放试验(LDH法)检测20％NHS对NPSCs及SV40-PED细胞的杀伤；MTT法检测两种细胞在20％NHS中培养时的活性；细胞免疫荧光检测补体C3c、C4d在NPSCs、SV40-PED的沉积；细胞免疫组化及免疫荧光检测补体攻膜复合物C5b-9在2种细胞上的沉积。Western blot检测并比较2种细胞clusterin的表达；RT-PCR检测并比较两种细胞补体调节蛋白CD46、CD59的表达。
【结果】NPSCs能够表达α-Gal，但明显弱于SV40-PED(平均荧光强度分别为41．78±2．39和95．17±2．39，P＜0．05)；NPSCs虽可与人血清中天然抗体IgG、IgM结合，但其结合程度明显弱于SV40-PED；20％NHS对NPSCs、SV40-PED的杀伤率分别为24．38％±0．50％和53．13％±14．53％(P＜0．01)；2种细胞在20％NHS中的活性分别为：98．73％±18．84％和52．43％±8．08％(P＜0．01)；免疫荧光及组化可见SV40-PED细胞上分别有补体C3c、C4d和C5b-9的沉积；但NPSCs仅有C3c和C4d沉积，而未见C5b-9沉积；Western blot证明，clusterin在NPSCs的表达略强于SV40-PED；RT-PCR证实，NPSCs表达的CD59显著强于SV40-PED，而CD46在此两种细胞的表达无明显差异。
【结论】NPSCs可以显著抵御人血清中天然抗体介导的杀伤作用，其机制可能与NPSCs低表达α-Gal导致其与天然抗体结合较少并高表达clusterin及CD59等补体调节蛋白从而抑制补体攻膜复合物形成有关。
PartⅠNeonatal 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.1.5×10~6 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±1.75×10~7 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.
PartⅡIsolation 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 Ca~(2+) 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.
PartⅢCotransplantation 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 1.5×10~6NPSCs were implanted under theleft renal capsule of recipient.Group 3 (n=8),1500 IEQ SD islets with 1.0×10~7NPSCs 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 1.5×10~6NPSCs,mean graft survival times were8.33±0.58 days,while co-transplanted with 1.0×10~7NPSCs,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 1.0×10~7NPSCs 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.
PartⅣResistance 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.
引文
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