细胞毒T淋巴细胞A4Ig抑制大鼠胰腺移植急性排斥反应的研究

B7／CTLA4-CD28是其中两大经典途径之一，我们试图利用细胞毒T淋巴细胞（CTL）A4Ig竞争性阻断B7／CD28通路，探讨其对大鼠胰腺移植急性排斥反应的作用及机制。     

中药移植合剂对大鼠胰腺移植急性排斥反应的作用

目的:观察大鼠胰腺移植后的细胞凋亡情况,以及中药移植合剂在急性排斥反应中的作用并探讨其作用机制.方法:建立大鼠全胰十二指肠移植模型,均为异基因移植(以SD、Wistar大鼠作为供、受体),共分5组,每组10只.第1组:阴性对照组;第2组:环孢素A (cyclosporin A, CsA)组,5 mg/(Kg·d);第3组:中药移植合剂组,每100 g体重1 mL/d;第4组:中西医结合组,中药移植合剂每100 g体重1 mL/d CsA 2.5 mg/(Kg·d);第5组:亚治疗剂量CsA组,2.5 mg/(Kg·d).术后第7 d每组处死5只大鼠,取移植胰腺,受体肝脏、肾脏行病理学检查,应用原位末端标记法(TUNEL)检测细胞凋亡情况.余鼠待出现急性排斥反应时处死,观察各组移植胰腺有功能存活时间.结果:中药移植合剂可以延长移植胰腺有功能存活时间,抑制胰腺腺泡细胞凋亡,其与亚治疗剂量CsA合用后可以达到治疗剂量CsA的抗排斥反应效果.结论:中药移植合剂具有一定的抗大鼠胰腺移植急性排斥反应的作用,作用机制与其抑制移植胰腺腺泡细胞凋亡有关,且与CsA具有协同作用.     

腺泡细胞凋亡在大鼠胰腺移植急性排斥反应中的意义

目的：观察移植胰腺的腺泡细胞凋亡及其与急性排斥反应的关系。方法：选用SD和Wistar大鼠进行全胰十二指移植。实验分为同基因移植组（Wistar→Wistar）和异基因移植组（SD→Wistar）两组。于术后第3d、5d和7d分批处死受体，取移植胰腺标本用HE染色和原位末端标记（TUNEL）技术检测移植胰腺切片，进行排斥反应的病理学评分和计数凋亡指数（AI）。结果：发生凋亡的细胞主要是腺泡细胞，同基因移植组胰腺有散在的腺泡细胞凋亡，AI在术后无明显变化。异基因移植组胰腺腺泡细胞凋亡在术后第3d、5d和7d逐渐升高，AI与急性排斥反应的病理学评分成正相关。结论：细胞凋亡与移植胰腺急性排斥反应的严重程度显著相关，凋亡指数可作为判断移植物损伤程度的指标，对急性排斥反应的诊断有一定的参考价值。     

PD-L1.Ig与1,25(OH)2D3联合应用对异基因大鼠胰腺移植后急性排斥反应的影响

目的 探讨PD-L1.Ig联合1,25(OH)2D3对大鼠胰腺移植急性排斥反应的免疫调控作用以及对大鼠移植胰腺存活时间的影响.方法 以F344大鼠为供体,Lewis大鼠为受体,建立4组原位胰腺移植模型.每组12例,共48例.A组:对照组、B组:PD-L1.Ig组、C组:1,25(OH)2D3组、D组:PD-L1.Ig+1,25(OH)2D3组;观察术后各组血糖变化,移植物存活以及组织病理学改变;流式细胞检测受体血、脾以及移植胰CD4+T细胞、CD8+T细胞、CD4+CD25+T细胞表达水平;酶联免疫吸附试验(ELISA)检测移植物局部白细胞介素(IL)-2、-4、-10、-12表达水平;于术后第7天分别杀死各组受体和供体鼠各2只,取受体脾细胞与供体作混合淋巴细胞反应(MLR).结果 与A比较,PD-L1.Ig组并未显著延长移植胰腺存活时间(P＞0.05),C、D组对改善血糖水平和延长移植胰腺存活时间作用明显(P＜0.01),其中,D组移植胰腺存活时间最长(23.9±0.8)d;与A组比较,B组无明显改善,C组排斥反应较A组明显减弱,而D组几乎未发生排斥反应;CD3+CD8+T细胞计数D、C、B与A组比较均有减少,差异有统计学意义(P＜0.01).CD4+T细胞D、C、B与A组的差异有统计学意义(P＜0.05).CD4+CD25+调节性T细胞A、B、C、D组逐渐升高,差异有统计学意义(P＜0.01),且以D组较为明显(C组比A组P＜0.01);D组明显抑制移植物局部Th1型细胞因子IL-2、IL-12的产生,显著提高Th2型细胞因子IL-4、10的水平;与对照组比较,各治疗组受体T淋巴细胞在MLR中表现对供体淋巴细胞特异性低反应性,能有效抑制T细胞对同种异体抗原的反应.结论 共刺激阻断剂PD-L1.Ig联合1,25(OH)2D3可有效抑制细胞免疫应答,干预急性排斥反应,显著延长大鼠移植胰腺存活时间.     

细胞凋亡在大鼠胰腺移植急性排斥反应中的作用

目的探讨细胞凋亡和相关因子Fas、FasL、bcl-2和bax在大鼠胰腺移植急性排斥反应中的作用,评价十二指肠黏膜活检在胰腺移植中诊断排斥反应的价值。方法选SD和Wistar大鼠行全胰十二指肠移植,实验分同基因胰腺移植组和异基因胰腺移植组。于移植术后第3、5及7d分批处死受体,取移植胰腺和十二指肠标本用HE染色和原位末端脱氧核糖核苷酸转移酶标记技术(TUNEL)检测移植物。免疫组化法检测移植后胰腺和十二指肠Fas、FasL、bcl-2和bax的表达。结果异基因胰腺移植组胰腺和十二指肠病理评分相同者占61.1%(11/18);评分不同者占38.9%(7/18),其中胰腺评分较高者6例,十二指肠黏膜评分较高者仅1例。异基因胰腺移植组胰腺和十二指肠病理学评分和细胞凋亡指数均明显高于同基因胰腺移植组(P<0.05,P<0.01)。胰腺和十二指肠细胞凋亡指数与急性排斥反应的病理学评分成正相关(r=0.965,P<0.01;r=0.942,P<0.01)。随着术后时间延长,排斥反应分级上升,细胞凋亡增加,FasL表达升高,在异基因移植组bcl-2表达下降,而Fas和bax表达无明显变化。结论细胞凋亡与移植胰腺急性排斥反应的严重程度呈正相关,细胞凋亡指数可作为判断移植物损伤程度的指标。FasL和bcl-2与胰腺移植急性排斥及组织损伤密切相关。十二指肠黏膜活检有助于判断有无胰腺急性排斥反应发生。     

胰腺移植急性排斥反应中的细胞凋亡

目的　观察胰腺移植急性排斥反应中的细胞凋亡变化。方法　对封闭群大鼠作单纯胰腺移植 (A组 )和胰脾联合移植 (B组 ) ,手术后第 3、7、12、2 0天每组各处死 5只 ,切取移植胰 ,石蜡包埋切片 ,原位DNA缺口末端标记 (TUNEL)法测定胰腺组织细胞凋亡。结果　A组术后第 3、7、12、2 0天胰腺细胞凋亡数分别为 (15 2 .5 5± 2 7.2 4)、(5 0 8.19± 117.83)、(70 1.33± 130 .17)和 (10 10 .5 6±114.6 9)个 /高倍视野 ;B组分别为 (12 4.87± 19.11)、(198.6 8± 31.99)、(492 .2 5± 93.0 1)和(6 49.80± 140 .91)个 /高倍视野。 2组各时点均显著高于正常胰腺。第 7、12、2 0天A组也显著高于B组。结论　胰腺移植急性排斥时细胞凋亡活性明显增高。     

大鼠小肠移植后外周血T淋巴细胞亚群的变化

利用免疫荧光染色技术及流式细胞仪对大鼠小肠移植后外周血Ｔ淋巴细胞亚群变化进行连续监测，以探讨细胞免疫功能变化在排斥反应中的意义。结果表明，排斥反应时首先出现ＣＤ４阳性细胞显著增高，随后出现ＣＤ８阳性细胞明显下降及ＣＤ４阳性细胞／ＣＤ８阳性细胞比值增高，在排斥反应后期其比值下降；使用环孢素Ａ作免疫抑制的大鼠Ｔ淋巴细胞各亚群均无显著性变化。本实验证明ＣＤ４阳性细胞及ＣＤ８阳性细胞共同参与了排斥反应，根     

大鼠小肠移植要后浸润移植小肠的T细胞动态研究

以远交系大鼠制作异位小肠移植模型，利用图像分析技术对浸润移植小肠的Ｔ淋巴细胞亚群进行连续定量测定，并与终期组织病理检查做比较，分析Ｔ淋巴细胞在排斥反应中的作用。结果表明未使用免疫抑制治疗的大鼠在术后其移植物均出现ＯＸ８＋细胞浸润增多，而使用环孢素Ａ的大鼠其ＯＸ８＋和Ｗ３／１３＋细胞浸润逐渐减少；术后７－１０天各组大鼠小肠移植物Ｗ３／２５＋细胞均呈上升趋势。本研究说明ＯＸ８＋细胞亚群在排斥反应中起重     

细胞毒T淋巴细胞在异种肝移植急性排斥反应中的作用

目的：探讨异种肝移植中细胞毒T淋巴细胞（CTL）参与急性排斥反应的机制。方法：三袖套法建立仓鼠到大鼠原位肝移植模型；免疫组化方法检测同基因组与异种移植组（XT组）移植肝术后CD8和CD4型淋巴细胞浸润及Fas-L，穿孔素的表达情况，并运用原位末端标记法检测移植肝细胞的凋亡情况。结果：XT组移植肝术后第2d汇管区出现淋巴细胞浸润，逐日增多，至术后5－6d达高峰；术后第4d穿孔素及Fas-L开始表达，术后第5－6d达高峰；穿孔素的表达始终高于Fas-L。结论：异种肝移植中T淋巴细胞参与了其免疫排斥反应，通过表达穿孔素或Fas-L使靶细胞坏死或凋亡。     

胰腺、肾脏在单独和联合移植中排斥反应的差异

目的 比较胰腺、肾脏在单独和联合移植中排斥反应的差异。方法 在大鼠同种异体单独胰腺移植、单独肾脏移植和肺肾联合移植基础上,对胰腺、肾脏在单独和联合移植中的排斥瓜反应分别进行了比较。结果 （１）胰腺在联合肾脏移植中受到肾脏保护,与单独上比,功能与形态不者到显著改观;（２）肾脏在联合胰腺移植时未受到胰腺保护,与单独移植比较,各项指标未得到显著改善。结论 胰腺与肾脏联合移植的结局优于单独胰腺移植,而肾脏     

联合阻断CD28和可诱导共刺激分子对小鼠同种异体胰岛移植物急性排斥反应的影响

目的 在小鼠同种异体胰岛移植模型上，通过联合阻断CD28和可诱导共刺激分子（ICOS），观察CTLA-4Ig及ICOS单克隆抗体（ICOSmAb）对胰岛移植物功能的影响。方法将小鼠随机分成4组，每组10只。联合阻断组：分别于移植后0．2、4d和1、3、5d腹腔内注射CTLA-4Ig50μg／kg和ICOSmAb100μg／kg体重组。ICOS组：移植后1、3．5d腹腔内注射ICOSmAb100μg/kg体重组。CTLA4组：移植后0、2、4d腹腔内注射CTLA-4Ig50μg/kg体重组。对照组：单纯胰岛移植，不做CT-LA-4Ig和ICOSmAb处理组。观察术后移植物存活时间和病理改变，RT-PCR检测移植组织中IL-2、IL-10mRNA表达，流式细胞仪检测CD4^＋、CD8^＋T淋巴细胞表达。结果 联合阻断组小鼠移植物存活时间平均为（31．00±6．57）d，较其他3组延长，差异有统计学意义（P〈0．05）；IL-2和IL-10mRNA表达水平分别为（44．23±6．24）和（65．23±11．02），与其他3组比较，IL-2mRNA表达差异有统计学意义（P〈0．05），而IL-10mRNA表达差异无统计学意义（P〉0．05）；CD4^＋、CD8^＋T淋巴细胞荧光阳性百分率分别为（13．73±0．49）％和（14．56±0．31）％，表达上调均不明显；移植胰岛光镜检查接近正常。结论 应用CTLA-4Ig和ICOSmAb联合阻断CD28和ICOS，可以明显抑制胰岛移植物排斥反应。     

Beneficial effects of costimulatory blockade with anti-inducible costimulator antibody in conjunction with CTLA4Ig on prevention of islet xenograft rejection from rat to mouse

BACKGROUND: Costimulatory signals have been reported to play an important role in islet-xenograft rejection, although the precise mechanisms remain unknown. The aim of the present study was to determine a role of a novel costimulatory molecule, inducible costimulator (ICOS), in rat islet-xenograft rejection in conjunction with CTLA4Ig with respect to cellular as well as humoral immune responses. METHODS: Isolated rat islets were transplanted into the liver of streptozotocin (180 mg/kg) induced diabetic mice. Cellular immune responses to islet xenografts, and productions of anti-rat antibody in mice were examined by flow cytometry (FACS) after transplantation. RESULTS: Intrahepatic rat islet xenografts were rejected in mice within 8 days after transplantation. FACS analysis revealed an expansion of CD8(+) T cells in the liver as well as a production of anti-rat antibody in recipient mice in association with rejection. The treatment with anti-ICOS antibody in conjunction with CTLA4Ig produced a marked prolongation of islet-xenograft survival with neither expansion of CD8(+) T cells nor production of anti-rat antibody, whereas, in contrast, those treated with anti-ICOS antibody or CTLA4Ig alone did not have prolonged survival, and CD8(+) T cells were expanded. CONCLUSION: These findings demonstrate that cellular rather than humoral immune responses are considered responsible for islet-xenograft rejection from rat to mouse and that the blockade of costimulatory signals with anti-ICOS antibody in conjunction with CTLA4Ig has a favorable effect on prevention of islet xenograft rejection.     

联合应用CTLA-4Ig及抗ICOS单克隆抗体延长小鼠移植胰岛存活时间

目的探讨共刺激信号阻断剂细胞毒T淋巴细胞相关抗原4免疫球蛋白（CTLA-4Ig）及抗共同刺激分子ICOS单克隆抗体（ICOSmAb）对移植胰岛功能的影响。方法以BALB／c小鼠为供者，C57BL／6糖尿病小鼠为受者，进行同种胰岛细胞移植。将移植后的小鼠随机分成4组，每组10只。ICOS组：移植后1、3、5d腹腔内注射ICOSmAb 100μg／kg；CTLA4组：移植后0、2、4d腹腔内注射CTLA-4Ig50μg／kg；联合阻断组：移植后腹腔注射CTLA-4Ig和ICOSmAb，用法同CTLA4组和ICOS组；对照组：单纯胰岛移植，不注射CTLA-4Ig和ICOSmAb。观察术后移植物存活时间和移植胰岛的病理改变；逆转录聚合酶链法（RT-PCR）检测移植胰岛组织中白细胞介素2（IL-2）、白细胞介素10（IL-10）mRNA的表达情况；应用流式细胞仪检测CD4^＋、CD8^＋T淋巴细胞表达情况。结果联合阻断组的小鼠移植胰岛存活时间较其它3组明显延长，移植胰岛的细胞形态经光镜检查接近正常。联合阻断组与其它3组比较，IL-2mRNA表达减少，差异有统计学意义（P〈0．05），IL-10mRNA的表达差异无统计学意义（P〉0．05）；移植术后21d，CD4^＋、CD8^＋T淋巴细胞表达上调不明显。结论应用CTLA-4Ig和ICOSmAb联合阻断CD28和共同刺激分子ICOS，可以明显的抑制排斥反应，延长移植胰岛的存活时间及存活率。     

Early diagnosis and treatment of pancreas allograft rejection

A major problem in vascularized pancreas transplantation is the lack of reliable methods for the early diagnosis and effective treatment of allograft rejection. Over a 2-year period, 54 rejection episodes occurred in 31 patients (13 isolated pancreas, 18 simultaneous pancreas-kidney recipients) with pancreaticoduodenocystostomy. A total of 253 radionuclide pancreas examinations were performed (mean 8.4 per patient) utilizing ^99mtechnetium-DTPA. Computer analysis generated a quantitative measure of blood flow to the allograft caused the technetium index (TI). Rejection episodes were characterized as isolated pancreas (22), combined pancreas-kidney (16), or isolated renal (16) allograft rejection in combined engraftments. The majority of rejection episodes occurred early (within 3 months of transplant, N=47) and were more responsive than late rejection to anti-rejection therapy (89.4% vs 42.9%, P=0.01). Mean urinary amylase (UA) levels and TI during normal allograft function were 29,398 U/l and 0.55%, while levels heralding rejection were 6,528 U/l and 0.40%, respectively (P<0.05). The treatment of rejection based upon renal dysfunction or combined renal and pancreas dysfunction resulted in significantly higher graft salvage with a lower incidence of hyperglycemia when compared to isolated pancreas allograft rejection. Of the 11 patients who developed hyperglycemia, 8 (72.7%) ultimately lost their pancreas grafts (P<0.001). Following therapy, a TI above 0.3% was associated with 97.4% graft survival, while levels below 0.3% resulted in a 70% rate of graft loss (P<0.001). Similarly, pancreas allografts with a UA above 10,000 U/l had 91.1% functional survival, while levels below 10,000 U/l resulted in a 66.7% rate of graft loss (P<0.001). Overall, reversal of rejection occurred in 83.3% of cases, with 9 grafts lost due to rejection at a mean of 4.7 months post-transplant. Therapy with ALG or OKT₃ was more effective in reversing allograft rejection than pulsed corticosteroids alone (68.8% vs 47.9%, P=0.05). Patient and pancreas allograft survival is 96.8% and 67.7%, respectively, after a mean follow-up interval of 14.9 months. Monitoring pancreas allograft function by UA, TI, and renal function (in simultaneous transplants) allows for the timely diagnosis and successful treatment of pancreas allograft rejection.     

猪全胰十二指肠移植排斥反应的早期诊断

目的 探讨移植胰腺分泌的胰液中肿瘤坏死因子（ＴＮＦ－α）水平、淀粉酶流量和受者的血糖水平在排斥反应早期诊断中的意义。方法 采用全胰切除的猪行全胰十二指肠移植，第１组受者不用免疫抑制剂；第２组受者应用甲泼尼龙＋雷公藤多甙片；第３组受者应用环孢素Ａ＋雷公藤多甙＋甲泼尼龙。观察受者的空腹血糖、胰液淀粉酶流量和ＮＴＦ－α水平。结果 第１组受者移植后第４ｄ胰液和淀粉酶流量下降，第８ｄ停止；第２组受者的胰液和     

胰腺腺泡细胞凋亡和Fas/Fas配体表达在大鼠胰腺移植急性排斥反应中的作用

目的探讨大鼠胰腺移植后细胞凋亡的变化、Fas/Fas配体(FasL)的表达及其与急性排斥反应的关系。方法实验分为同基因移植组和异基因移植组。分别于术后第3、5、7天处死受体,取移植胰腺,应用原位末端标记法(TUNEL)检测细胞凋亡,计算凋亡指数(AI),应用免疫组织化学及Westernblot检测Fas/FasL的表达。结果细胞凋亡主要发生在胰腺腺泡细胞,同基因移植组有散在的腺泡细胞凋亡,AI在术后无明显变化,Fas/FasL表达阴性;异基因移植组在术后第3、5、7天胰腺腺泡细胞凋亡发生率逐渐增加(28.40±3.75,39.40±5.59,57.30±8.53,P<0.01),Fas/FasL表达逐渐增强,AI与急性排斥反应程度呈显著正相关(rs=0.932,P<0.01)。结论胰腺腺泡细胞凋亡和Fas/FasL表达在胰腺移植急性排斥反应中发挥重要作用,凋亡指数对胰腺移植急性排斥反应的诊断有一定的参考价值。     

Primed allospecific T cells prevent the effects of costimulatory blockade on prolonged cardiac allograft survival in mice.

Costimulatory blockade can induce long‐term allograft survival in naïve animals, but may not be as effective in animals with previously primed immune repertoires. We attempted to induce long‐term graft survival in B10.D2 recipients of B10.A cardiac allografts using donor‐specific transfusion (DST) plus anti‐CD40 ligand antibody (αCD40L). Recipients were either naïve mice, or mice previously primed to B10.A or third party alloantigens through engraftment and rejection of skin transplants. Untreated naïve mice rejected cardiac transplants by day 15 and contained a high frequency of primed, donor‐reactive T cells. Donor‐specific transfusion/αCD40L treatment of naïve animals induced long‐term graft survival associated with low frequencies of donor‐reactive T cells. Previous priming of donor‐specific T cells through rejection of B10.A, but not third party, skin grafts prevented the effects of DST/αCD40L on prolonging survival of B10.A hearts. Moreover, adoptive transfer of CD3+, CD4+ or CD8+ T cells from B10.A skin‐graft‐primed animals prevented the effects of DST/αCD40L. The data demonstrate that animals with immune repertoires containing previously primed, donor‐reactive T cells are resistant to the effects of costimulatory blockade. The findings have important implications for ongoing, costimulatory blockade‐based trials in humans, whose T‐cell repertoires are known to contain memory alloreactive T cells.     

Histological features of acute pancreatic allograft rejection after pancreaticoduodenal transplantation in the rat

For characterization of histopathological changes during pancreas graft rejection, pancreaticoduodenal transplants were performed in three groups: (1) Brown Norway into diabetic Lewis rats without immunosuppression, (2) Brown Norway into diabetic Lewis rats with cyclosporin A, and (3) Lewis into Lewis rats. Diffuse inflammatory infiltration of the acini by mononuclear cells indicated the onset of rejection (stage I). Shortly after acinar infiltration, damage to small and large interlobular excretion ducts occurred. This took the form of florid circumferential inflammation and vacuolar degeneration of epithelium similar to the bile duct damage seen in primary biliary cirrhosis, graft-versus-host disease, and liver allograft rejection (stage II). Thereafter, endothelialitis and destruction of islets were evident, consistent with a more advanced and irreversible stage of rejection (stage III). Acinar inflammation and moderate duct lesions were not prevented by immunosuppression but were delayed. Nonetheless, severe vascular changes and loss of islets were avoided. We conclude that duct lesions are a reliable criterion for pancreas allograft rejection. They are more sensitive than vascular changes and more specific than cellular infiltration of acinar tissue, which may also occur in infection.     

Histological features of acute pancreatic allograft rejection after pancreaticoduodenal transplantation in the rat

Abstract. For characterization of histopathological changes during pancreas graft rejection, pancreaticoduodenal transplants were performed in three groups: (1) Brown Norway into diabetic Lewis rats without immunosuppression, (2) Brown Norway into diabetic Lewis rats with cyclosporin A, and (3) Lewis into Lewis rats. Diffuse inflammatory infiltration of the acini by mononuclear cells indicated the onset of rejection (stage I). Shortly after acinar infiltration, damage to small and large interlobular excretion ducts occurred. This took the form of florid circumferential inflammation and vacuolar degeneration of epithelium similar to the bile duct damage seen in primary biliary cirrhosis, graft-versus-host disease, and liver allograft rejection (stage II). Thereafter, endothelialitis and destruction of islets were evident, consistent with a more advanced and irreversible stage of rejection (stage III). Acinar inflammation and moderate duct lesions were not prevented by immunosuppression but were delayed. Nonetheless, severe vascular changes and loss of islets were avoided. We conclude that duct lesions are a reliable criterion for pancreas allograft rejection. They are more sensitive than vascular changes and more specific than cellular infiltration of acinar tissue, which may also occur in infection.