Sertoli细胞与成人胰岛细胞共同培养的研究

目的观察塞尔托利（Sertoli）细胞对体外培养的成人胰岛细胞形态、存活率及功能的影响。方法胰腺、睾丸取自志愿捐赠的成年男性尸体多器官供者,共12例。分离纯化后的成人胰岛细胞分为单独培养组和共同培养组,单独培养组取成人胰岛细胞单独培养,共同培养组为成人胰岛细胞＋Sertoli细胞共同培养,均在RPMI1640培养液培养14d,采用倒置相差显微镜观察胰岛细胞形态,比较两组的胰岛细胞存活率、胰岛素分泌量和胰岛素刺激指数。结果培养14d后,共同培养组胰岛细胞存活率为（90±3）%,较单独培养组的（57±4）%明显提高（P〈0.01）,胰岛细胞的形态亦较单独培养组完整。共同培养组胰岛细胞始终对葡萄糖刺激保持较高的敏感度,而单独培养组胰岛细胞对葡萄糖刺激的敏感度随时间的延长明显降低（P〈0.05）。培养14d后,共同培养组的胰岛素分泌量为（249±12）mIU/L、胰岛素刺激指数为8.15±0.64,而单独培养组则分别为（47±7）mIU/L和1.68±0.34,两组比较差异有统计学意义（均为P〈0.01）。结论成人胰岛细胞与Sertoli细胞共同培养可以提高胰岛细胞的存活率,改善胰岛细胞的功能。     

大鼠胰岛细胞转染Wee1Hu基因延长异种胰岛细胞移植后存活时间

目的 探讨大鼠胰岛细胞转染Wee1Hu基因对异种胰岛细胞移植后存活时间的影响。方法 以Wistar大鼠为供者，Balb／c糖尿病小鼠为受者，进行异种胰岛细胞移植。实验分为2组，每组20只。实验组：将转染Wee1Hu基因的1×10^6个供者胰岛细胞悬于0．5ml无菌生理盐水中，注射至受者的腹腔；空白对照组：将1×10^6个空载体胰岛细胞用与实验组相同的方法注射至受者腹腔。实验组和空白对照组的部分受者在移植前1周腹腔注射降植烷0．5ml，并于胰岛细胞移植后开始口服环孢素A30mg／kg，直至实验结束。监测2组胰岛细胞移植后的胰岛素释放功能及存活时间。结果 胰岛细胞移植后，空白对照组的受者均维持高血糖，且体重持续下降，平均生存时间为（18±2．5）d；实验组的受者血糖在2d内均降至正常，且体重增加，生存时间延长（38±3．5）d；两组相比较，差异有统计学意义（P〈0．05）。空白对照组维持正常血糖时间为（8±2．3）d，而实验组为（10±2．5）d。实验组中采用免疫抑制剂治疗的受者，长期维持正常血糖，平均维持时间超过30d，与空白对照组中采用免疫抑制剂治疗的受者比较，差异有统计学意义（P〈0．05）。结论 大鼠胰岛细胞转染Wee1Hu基因可延长异种胰岛细胞移植后的存活时间，与免疫抑制剂协同作用时效果更佳。     

诱导供者胰岛细胞高表达血红素加氧酶-1延长大鼠胰岛移植物的存活时间

目的 探讨钴原卟啉(CoPP)诱导大鼠胰岛细胞高表达血红素加氧酚1(HO-1)后,对延长胰岛移植物存活时间的作用.方法 (1)将分离和纯化的供者(BN大鼠)胰岛细胞分为CoPP诱导组和未诱导组.CoPP诱导组供者在分离胰岛细胞前3天和前1天腹腔注射2.5 mg/kg的CoPP,未诱导组不注射CoPP.诱导后,采用免疫荧光法及Western免疫印迹法检测两组胰岛细胞中HO-1的表达情况,采用酶联免疫吸附试验(ELISA)和葡萄糖刺激试验检测胰岛细胞的胰岛素释放水平.(2)Lewis大鼠经四氧嘧啶静脉注射后建立糖尿病模型,取10只成功建立糖尿病模型的大鼠作为胰岛细胞移植的受者,随机平均将受者分为实验组和对照组,分别移植经CoPP诱导和未经诱导的供者胰岛细胞.移植后,观察和比较两组受者胰岛移植物的存活时间和发生排斥反应后胰岛移植物的组织病理学变化.结果 CoPP诱导组胰岛细胞高表达HO-1,而未诱导组不表达HO-1;CoPP诱导组和未诱导组供者胰岛细胞胰岛素分泌量,在低糖刺激下分别为(15.65±0.89)mU/L和(12.28±0.89)mU/L(P>0.05),在高糖刺激下分别为(46.60±1.13)mU/L和(19.01±1.49)mU/L(P<0.05),刺激指数分别为2.98±0.10和1.55±0.01(P<0.05).实验组和对照组胰岛移植物平均存活时间分别为(12.20±5.67)d和(5.60±1.14)d(P<0.05);当受者发牛排斥反应时,对照组胰岛移植物周边可见明显的淋巴细胞、成纤维细胞以及单个核细胞浸润,而实验组细胞浸润的程度明显较轻.结论 CoPP可诱导大鼠胰岛细胞高表达HO-1,其对胰岛细胞有明显保护作用.移植高表达HO-1的胰岛细胞能显著延长胰岛移植物的存活时间.     

Fas配体阳性睾丸细胞对共移植的胰岛细胞存活起全身和/或局部的免疫豁免作用

目的探讨Fas配体阳性睾丸细胞对共移植的胰岛细胞存活起全身和/或局部的免疫豁免作用。方法将同种大鼠胰岛细胞与不同数量的睾丸细胞同侧或两侧共移植于糖尿病受体肾包膜下,观察移植物功能和存活情况,以及移植物内淋巴细胞凋亡。结果单纯移植胰岛组(对照组)平均存活期为(4.6±1.1)天。睾丸细胞和胰岛细胞同侧共移植时,当睾丸细胞数为1×106(A组)时,胰岛细胞平均存活期为(23.8±4.6)天;睾丸细胞数增至1×107时(B组),胰岛细胞存活期大于(57.5±4.0)天,均较对照组明显延长(P<0.01)。两侧共移植时,当睾丸细胞数为1×105(C组),胰岛细胞平均存活期为(6.0±1.4)天,与对照组相似(P>0.05);睾丸细胞数增至1×106(D组),胰岛细胞存活期(11.5±3.1)天较对照组或C组延长(P<0.01);睾丸细胞数增至1×107(E组)时,胰岛细胞存活期(31.2±15.9)天延长更加明显(P<0.01)。相同剂量的睾丸细胞与胰岛细胞分别同侧和两侧共移植时发生移植排斥反应各自的病理表现是不同的,前者移植物内有淋巴细胞浸润,部分胰岛玻璃样变,抗胰岛素抗体染色仅见少量阳性细胞,而后者胰岛细胞移植物内有大量的淋巴细胞和少量的中性粒细胞浸润,抗胰岛素抗体阳性细胞较少,未见胰岛玻璃样变。原位细胞凋亡检测发现同侧共移植后的移植物与肾实质交界处有淋巴细胞凋亡。电镜检查发现胰岛移植物存活超过60d的标本可见大量存活的胰岛细胞团和睾丸Sertoli细胞,有散在的凋亡淋巴细胞。结论睾丸细胞与同种胰岛细胞移植后不仅可诱导局部免疫豁免,而且也具有一定的全身免疫保护作用,但较局部免疫豁免作用弱;这些作用可延长移植物的存活期,并具有剂量依赖性。     

CTLA4Ig基因在延长大鼠移植胰岛存活中的作用

胰岛移植是治疗糖尿病的理想方法 ,有效的胰岛移植可代替长期胰岛素的使用。胰岛移植存在的最主要问题是免疫排斥反应 ,因此降低移植物免疫原性和诱导受体对胰岛移植物耐受是解决排斥反应问题的方法。ＣＴＬＡ4Ｉｇ是阻断Ｂ7 ＣＤ2 8共刺激信号传导最有效的免疫抑制剂 ,国外在这方面研究较多 ,取得了良好效果[1] ,国内尚未见报告。本研究利用脂质体 (ｌｉｐｏｆｅｃｔｉｎ)载体包裹ＣＴＬＡ4ＩｇｃＤＮＡ质粒后转染鼠胰岛和肌肉细胞 ,观察ＣＴＬＡ4Ｉｇ基因表达在延长糖尿病大鼠移植胰岛存活方面的作用。一、材料和方法1 动物和主要试剂 :大…     

小鼠自体肝脏星状细胞联合同种异体胰岛细胞移植延长移植物存活时间的研究

目的 研究小鼠自体肝脏星状细胞联合同种异体胰岛细胞移植的新方法对胰岛移植物存活时间的作用.方法 选择雄性BALB/c小鼠为胰岛移植模型的供者,雄性C57BL/6糖尿病小鼠为受者.随机将受者分为A、B两组.A组:仅采用供者的胰岛细胞移植;B组:采用受者的肝脏星状细胞(HSCs)与供者胰岛细胞混合后共同移植.术后定期测定受者尾静脉血的血糖含量.结果 B组受者胰岛移植物的存活时间明显延长,血糖含量维持正常的中位时间为66 d(30～180 d),而A组血糖含量维持正常的中位时间为11 d(9～15 d),两组比较,差异有统计学意义(P＜0.001).结论 受者肝脏星状细胞能延长共同移植的同种异体胰岛移植物存活时间.     

激活型Akt1转染大鼠胰岛联合应用细胞毒T淋巴细胞相关抗原4免疫球蛋白延长异种胰岛移植存活

目的 观察激活型Akt1基因转染大鼠胰岛对异种胰岛移植功能和存活的影响.方法 提取大鼠胰岛培养转染,AO/EB和Tunel染色检测存活和凋亡;BALB/C糖尿病小鼠肾被膜下胰岛移植.分4组,实验组:Akt1转染胰岛联合应用细胞毒T淋巴细胞相关抗原4免疫球蛋白(CTLA-4Ig);Akt1组:移植Akt1转染的胰岛;未转染组:单纯胰岛移植,CTLA-4Ig组:单纯胰岛联合应用CTLA-4Ig;观察胰岛移植后功能存活时间和平均生存时间.结果 Akt1转染胰岛体外细胞抗凋亡生存率提高了25%;实验组移植物功能存活(32.5±6.6)d,平均生存时间(39.6±5.9)d比Akt1组(15.4±3.5)、(22.5±1.7)d和CTLA-4Ig组(18.7±4.5)、(21.7±3.5)d未转染组(4.5±2.8)、(6.8±3.1)d明显延长(P<0.05).结论 激活型Akt1转染大鼠胰岛联合应用CTLA-4Ig,可减少移植胰岛凋亡,延长异种胰岛移植物存活时间.     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

目的 观察小鼠Sertoli细胞是否能在异体内起到诱导局部免疫耐受、保护共移植异体胰岛的作用.方法 以糖尿病C57小鼠作移植受体,随机分4组,每组6只;以正常BALB/C小鼠为胰岛供体,正常C57小鼠和正常BALB/C小鼠各作为Serloli细胞供体.A组:单纯移植异体胰岛;B组:移植来源于C57小鼠的Sertoli细胞+BALB/C小鼠来源的胰岛;C组:移植均来源于BALB/C小鼠的Sertoli细胞及胰岛;D组:假手术组.监测各组移植受体的血糖尿糖变化,观察移植物的存活时间.结果 A组移植物平均存活时间为(6.50±2.35)d;B组为(55.67±4.84)d;C组为(51.33±5.05)d;D组未观察到血糖正常.B组及C组的移植方式均可逆转糖尿病小鼠的高血糖状态,移植物存活期均较A组有明显延长,其差异有统计学意义(P<0.05);而B组与C组的移植物存活时间差异无统计学意义(P>0.05).结论 同种异体来源的睾丸Sertoli细胞在异体内可起到诱导局部免疫耐受的效果,对共移植同种异体胰岛起到保护作用,其效果与自体睾丸Sertoli细胞相当.     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

Objective To study whether the sertoli cell allograft can achieve the immunotolerance and protect the co-transplant islet allograft on the heterotopic situation. Methods The diabetic C57 mice were used as recipients, and healthy BALB/C mice as islet donors,respectively. Healthy BALB/C and C57 mice were used as testis sertoli cell donors. The recipients were randomly divided into 4 groups,6 mice in each group : group A: only transplant with islet allograft;group B: co-transplant with islet allograft and serto-li isograft;group C:co-transplant with islet allograft and sertoli allograft;group D:sham-operated group. The blood and urine glucose levels in the models, and the survival time of the graft were observed. Results The mean survive time of graft in groups A, B, and C was (6.50±2.35 ), (55.67±4.84), and (51.33± 5.05 ) days respectively. In group D, blood glucose level was abnormal. The hyperglycemia of the diabetic C57 mice could be reversed by the transplant methods of groups B and C. The mean survival time in groups B and C was longer than in group A P < 0.05, but there was no significant differences between groups B and C,P > 0.05. Conclusion The sertoli cells can induce local immunotolerance and protect the co-transplant islet allograft. Sertoli cell isograft can obtain the same local immunotolerance as the sertoli cell allograft.     

小鼠异体Sertoli细胞诱导胰岛移植免疫耐受的作用

Objective To study whether the sertoli cell allograft can achieve the immunotolerance and protect the co-transplant islet allograft on the heterotopic situation. Methods The diabetic C57 mice were used as recipients, and healthy BALB/C mice as islet donors,respectively. Healthy BALB/C and C57 mice were used as testis sertoli cell donors. The recipients were randomly divided into 4 groups,6 mice in each group : group A: only transplant with islet allograft;group B: co-transplant with islet allograft and serto-li isograft;group C:co-transplant with islet allograft and sertoli allograft;group D:sham-operated group. The blood and urine glucose levels in the models, and the survival time of the graft were observed. Results The mean survive time of graft in groups A, B, and C was (6.50±2.35 ), (55.67±4.84), and (51.33± 5.05 ) days respectively. In group D, blood glucose level was abnormal. The hyperglycemia of the diabetic C57 mice could be reversed by the transplant methods of groups B and C. The mean survival time in groups B and C was longer than in group A P < 0.05, but there was no significant differences between groups B and C,P > 0.05. Conclusion The sertoli cells can induce local immunotolerance and protect the co-transplant islet allograft. Sertoli cell isograft can obtain the same local immunotolerance as the sertoli cell allograft.     

Long-term survival of neonatal porcine Sertoli cells in non-immunosuppressed rats

Abstract: Sertoli cells from the testis contain immunoprotective properties which allow them to survive as allografts and also to protect islets and adrenal chromafin cells from immune rejection without the use of immunosuppressive drugs. Experiments were designed to determine whether xenogeneic neonatal porcine Sertoli cells (NPSCs) survive transplantation in rats without the use of immunosuppression. NPSCs (92.2  ±  5.1%) were isolated, cultured and then transplanted under the kidney capsule of non-immunosuppressed Lewis rats. To assess survival, grafts were removed after 4, 20, 30, 40, 60, and 90 days post-transplant and immunostained for the Sertoli cell marker vimentin. Survival was confirmed by polymerase chain reaction (PCR) for the porcine mitochondrial cytochrome oxidase II (COII) subunit gene, a marker for porcine tissue. In both methods, NPSCs were detected in the grafts for at least 90 days. Histologically, NPSCs were clustered in small aggregates or organized in tubule-like structures. When stained for the presence of proliferating cell nuclear antigen (PCNA), many Sertoli cells stained positive at 20 days post-transplant, indicating not only cell survival but also Sertoli cell proliferation. The number of PCNA postive cells decreased somewhat by 40 days with almost no positive Sertoli cells at 60 and 90 days. These data demonstrate that NPSCs survive long-term following xenotransplantation in rats, which to our knowledge is the first report of a discordant xenograft surviving without immunosuppression in a non-immunoprivileged site. Further study of the mechanism of NPSC xenograft survival may provide clues for promoting a local tolerogenic environment.     

Prolongation of islet allograft survival.

Pretreatment of donor rats with irradiation and silica followed by in vitro culture of the islets for 1 to 2 days prolonged survival of allografts across a minor histocompatibility barrier if "hand-picked," clean islets were used for transplantation. Pretreatment of donor rats with irradiation and silica in conjunction with a single injection of antilymphocyte serum (ALS) into the recipient produced a prolongation of survival of hand-picked islets transplanted across a major histocompatibility barrier.     

转染IKK2dn的受者树突状细胞回输延长同种异体肾移植大鼠的存活时间

目的 探讨IKK2dn基因转染并负载供者抗原的受者未成熟树突状细胞(imDC)延长同种异体肾移植大鼠的存活时间及其机制.方法 获取和培养Lewis大鼠骨髓源性DC,转染IKK2dn并负载BN大鼠可溶性抗原进行体外实验,检测CD86和主要组织相容性复合物(MHC)Ⅱ的表达及DC刺激T淋巴细胞增殖的能力.肾移植受者为Lewis大鼠,用随即数字表法分DC组、空转染组、转染组、对照组,术前7d分别输注1×10~7个D、Adv-0-DC、负载BN抗原的Adv-IKK2dn-DC和等量生理盐水,供者均为BN大鼠.另设第三方供者组,术前处理同转染组,供者为Wistar大鼠.移植后检测各组受者T淋巴细胞的增殖能力及血清白细胞介素2(IL-2)和γ干扰素(IFN-γ)的表达水平,观察各组大鼠的存活时间和发生排斥反应情况.结果 DC的体外实验结果显示:与转染IKK2dn前相比,转染后DC仍能低水平表达CD86和MHC Ⅱ,负载供者抗原后CD86和MHCⅡ表达均增加,而转染IKK2dn后再负载供者抗原,CD86和MHC Ⅱ的表达未发生明显变化;DC负载供者抗原后,刺激T淋巴细胞增殖的能力明显增强(P<0.05),而转染IKK2dn并负载供者抗原后不能有效刺激T淋巴细胞增殖.肾移植术后的检测结果显示:转染组T淋巴细胞的增殖能力明显低于其他4组(P<0.05或P相似文献   

Prolongation of allograft survival by passenger donor regulatory T cells

Tissue resident lymphocytes are present within many organs, and are presumably transferred at transplantation, but their impact on host immunity is unclear. Here, we examine whether transferred donor natural regulatory CD4 T cells (nT‐regs) inhibit host alloimmunity and prolong allograft survival. Transfer of donor‐strain lymphocytes was first assessed by identifying circulating donor‐derived CD4 T cells in 21 consecutive human lung transplant recipients, with 3 patterns of chimerism apparent: transient, intermediate, and persistent (detectable for up to 6 weeks, 6 months, and beyond 1 year, respectively). The potential for transfer of donor nT‐regs was then confirmed by analysis of leukocyte filters recovered from ex vivo normothermic perfusion circuits of human kidneys retrieved for transplantation. Finally, in a murine model of cardiac allograft vasculopathy, depletion of donor CD4 nT‐regs before organ recovery resulted in markedly accelerated heart allograft rejection and augmented host effector antibody responses. Conversely, adoptive transfer or purified donor‐strain nT‐regs inhibited host humoral immunity and prolonged allograft survival, and more effectively so than following administration of recipient nT‐regs. In summary, following transplantation, passenger donor‐strain nT‐regs can inhibit host adaptive immune responses and prolong allograft survival. Isolated donor‐derived nT‐regs may hold potential as a cellular therapy to improve transplant outcomes.