激活型Akt1转染大鼠胰岛联合免疫抑制剂在异种胰岛移植中的应用

目的 探讨腺病毒载体介导激活性Akt1基因(Adv-CA-Akt1)转染大鼠胰岛对异种移植胰岛功能和存活的影响.方法 以BALB/C糖尿病小鼠为受体,分离纯化雄性Wistar大鼠胰岛,体外培养,Adv-CA-Akt1转染后异种胰岛移植.受体小鼠分3组,实验组:Adv-CA-Akt1转染的大鼠胰岛体外培养24 h,小鼠肾被膜下移植,并口服环孢素A(CsA)30 mg·kg-1·d-1;CsA组:未转染胰岛移植,同剂量环孢素口服;对照组:单纯胰岛移植.每只接受300胰岛当量(IEQs)移植.检测术后血糖,移植物存活时间及组织病理学.结果 实验组和CsA组术后2 d血糖即降至正常,胰岛功能存活时间分别为(21.0±3.65)d和(9.0±2.54)d,而对照组血糖短暂下降后再次升高,胰岛功能存活时间(4.2±2.6)d.实验组小鼠生存时间为(31.0±5.67)d比CsA组(17.0±3.35)d和对照组(10.0±1.52)d明显延长,三组比较差异有统计学意义(P<0.05);胰岛素免疫组化染色实验组.肾被膜下见较多有功能胰岛细胞团,而CsA组和对照组胰岛素染色阳性细胞数减少.结论 Adv-CA-Akt1转染大鼠胰岛联合应用免疫抑制剂,可提高胰岛功能,延长异种胰岛移植物存活时间.     

激活型Akt1转染大鼠胰岛对移植物凋亡和再血管化的影响

目的 探讨腺病毒介导激活型蛋白激酶B(Akt1)基因(Adv-CA-Akt1)转染大鼠胰岛对同种糖尿病大鼠胰岛移植物凋亡和再血管化的影响.方法 分离纯化Wistar大鼠胰岛,转染Adv-CA-Akt1.36只糖尿病Wistar大鼠完全随机均分为3组: Adv-CA-Akt1组(Adv-CA-Akt1转染的胰岛移植); Adv-LacZ组(Adv-LacZ转染的胰岛移植); 未转染组(单纯胰岛移植).术后每日测血糖,隔日测血清胰岛素浓度,术后10 d行静脉糖耐量试验(IVGTT)观察胰岛功能; HE染色和胰岛素免疫组化检测胰岛功能,细胞凋亡原位检测胰岛凋亡,CD31免疫组化计数微血管密度(MVD).结果 Adv-CA-Akt1组大鼠血糖术后2 d恢复正常,Adv-LacZ组和未转染组血糖虽下降但仍高于正常; Adv-CA-Akt1组术后各时相血清胰岛素水平明显高于其他2组(P＜0.05).IVGTT示Adv-CA-Akt1组血糖下降较快,60 min恢复至空腹水平; 另2组下降慢,60 min仍未恢复至空腹水平.术后3 d,Adv-CA-Akt1组肾被膜下存活胰岛细胞数量多,胰岛素免疫组化证明为有功能的胰岛.Adv-CA-Akt1组胰岛凋亡率比Adv-LacZ组和未转染组降低约25%; 术后12 d,Adv-CA-Akt1组MVD比Adv-LacZ组及未转染组明显增高(P＜0.05).结论 激活型Akt1转染大鼠胰岛能够抑制胰岛细胞凋亡,提高胰岛β细胞功能,促进移植物早期再血管化.     

基因转移CTLA4-Ig和抗CD154抗体在异种胰岛移植排斥反应中的作用

目的 探讨基因转移细胞毒性T细胞相关抗原4免疫球蛋白(CTLA4-Ig)和抗T细胞分化群154(CD154)抗体在异种胰岛移植排斥反应中的作用及机理.方法 建立人-大鼠异种胰岛移植模型,用携带CTLA4-Ig基因的重组腺病毒感染移植胰岛细胞,并用抗CD154抗体进行治疗,观察糖尿病大鼠胰岛移植后血糖变化、生存情况及移植物病理形态学改变,检测移植物CTLA4-Ig、胰岛素的表达和移植大鼠白细胞介素2(IL-2)、肿瘤坏死因子(TNF)-α的水平变化.结果 (1)糖尿病大鼠移植后2 d血糖降至正常,对照组血糖平均在移植后8 d升高,抗体治疗组、转染组和联合治疗组血糖分别在18、25和36 d升高.(2)对照组、抗体治疗组、转染组和联合治疗组的移植物存活时间分别为(10.0±2.1)d、(22.0±8.2)d、(28.0±6.5)d和(37.0±9.3)d,各组间比较差异有统计学意义(P＜0.05);移植大鼠生存时间分别为(21.0±5.7)d、(35.0±6.5)d、(48.0±8.5)d和(65.0 ±12.5)d,各组间比较差异有统计学意义(P＜0.05).(3)对照组在移植后1周内,IL-2、TNF-α的水平均急剧上升,较移植前显著升高(P＜0.01).(4)各治疗组移植物见成片的胰岛细胞团,未见淋巴细胞浸润,转染组和联合治疗组移植物可见CTLA4-Ig和胰岛素的表达.结论 基因转移CTLA4-Ig和抗CD154抗体均可抑制异种胰岛移植排斥反应,二者联合效果优于单独使用.     

转染细胞毒性T淋巴细胞相关抗原4免疫球蛋白基因的同供体大鼠树突状细胞对胰岛移植物存活的影响

目的　探讨转染细胞毒性T淋巴细胞相关抗原4(转染CTLA4Ig基因)的同供体大鼠DC细胞对同种大鼠胰岛移植的影响。方法　链尿菌素(STZ) 60mg/kg体重腹腔内注射制作SD大鼠糖尿病模型,胶原酶法分离、Ficoll 40 0密度梯度离心法纯化胰岛,GM CSF +IL 4诱生培育的方法,获得高纯度的DC ,含目的基因CTLA4Ig重组腺病毒AdvCTLA4Ig ,转染同供体DC细胞,与胰岛细胞同时移植于糖尿病受体大鼠肾包膜下,免疫组织化学、Dot ELISA、逆转录 聚合酶链反应(RT PCR)检测CTLA4Ig基因在实验组和对照组DC细胞中的表达,并检测受体大鼠的血糖浓度变化,同时观察受体存活情况。结果　实验组DC细胞有CTLA4Ig表达,而对照组DC细胞没有CTLA4Ig表达,实验组正常血糖维持时间(17.3±2 .4)d较对照组(10 .1±1.5 )d、空白对照组(8.3±1.2 )d显著延长,实验组、对照组和空白对照组受体大鼠存活天数分别为(3 4.5±3 .4)d、(14 .7±2 .3 )d和(11.2±1.4)d ,实验组高于对照组(P <0 .0 1)。结论　表达CTLA4Ig基因的DC细胞可能诱异胰岛移植免疫耐受,延长胰岛移植物的存活时间     

阻断糖尿病小鼠共刺激分子延长异种胰岛移植细胞功能

目的 阻断多种共刺激分子而诱导免疫耐受,以延长移植到糖尿病小鼠体内的猪胰岛细胞的功能.方法 将猪胰岛细胞移植于链脲佐菌素诱导的雄性C57BL/6糖尿病小鼠左肾被膜下,应用CTLA-4Ig融合蛋白、抗LFA-1抗体和抗CD40L抗体阻断多种其刺激分子,观察移植效果及移植物存活时间.结果 猪胰岛细胞移植后可降低糖尿病小鼠血糖;与对照组相比.实验组胰岛细胞存活时间明显延长,结论应用CTLA-4Ig融合蛋白、抗LFA-1抗体和抗CD40L抗体可延长异种移植胰岛细胞存活时间.     

新生猪Sertoli细胞与大鼠胰岛细胞联合移植延长大鼠移植胰岛的存活时间

目的 探讨新生猪Sertoli细胞(NPSCs)与大鼠胰岛细胞联合移植对延长大鼠同种异体胰岛移植物存活时间的作用及其主要的机制.方法 将糖尿病Wistar大鼠随机分为3组.(1)单纯移植组(n=6):单纯移植1500个胰岛当量(IEQ)的SD大鼠胰岛细胞至糖尿病大鼠的左肾包膜下;(2)分侧移植组(n=4):将1500个IEQ的SD大鼠胰岛细胞移植到糖尿病大鼠的左肾包膜下,同时将1×10~7个NPSCs移植到糖尿病大鼠的右肾包膜下;(3)混合移植组(n=6):将1500个IEQ的SD大鼠胰岛细胞和1×10~7个NPSCs混合移植到糖尿病大鼠的左肾包膜下.移植后每天监测各组的血糖水平,以了解移植物的存活时间;移植后发生排斥反应时获取移植物标本,行HE和免疫组织化学染色,观察移植物中CD3~+T淋巴细胞浸润情况及细胞凋亡调控基因(Bcl-2)和血红素氧合酶1(HO-1)基因的表达水平.结果 单纯移植组、分侧移植组和混合移植组胰岛移植物存活时间分别为(5.7±1.0)d、(5.3±0.5)d和(16.3±1.4)d,混合移植组存活时间较其他两组显著延长(P<0.05).单纯移植组移植区可见大量淋巴细胞浸润,主要为CD3+T淋巴细胞;混合移植组移植区Bcl-2基因呈高表达;各组移植区HO-1基因均有表达,差异不明显.结论 NPSCs与大鼠胰岛细胞联合移植可以延长大鼠同种异体胰岛移植物的存活时间,其机制可能与NPSCs抑制移植物淋巴细胞浸润、促进Bcl-2基因高表达的局部免疫调节作用有关.     

大鼠胰岛转染人血红素氧合酶-1基因对同种胰岛移植的影响

目的探讨大鼠胰岛转染人血红素氧合酶-1(HO-1)基因在胰岛移植中的潜在应用价值。方法采用携带人HO-1基因的腺病毒对新分离的SD大鼠胰岛细胞进行转染;对体外培养的胰岛细胞使用重组人肿瘤坏死因子α及放线菌酮诱导凋亡。使用流式细胞术检测凋亡率;每只链脲霉素诱导的糖尿病模型大鼠门静脉内置入约1200个胰岛当量的胰岛后,观察糖尿病大鼠血糖及体重变化;免疫组织化学法检测肝内移植胰岛细胞的胰岛素、HO-1蛋白表达及表达CD3抗原的淋巴细胞浸润情况。结果转染人HO-1基因的胰岛细胞凋亡率明显低于对照组(P<0.05);单纯胰岛细胞移植后移植物存活时间为(5.33±4.18)d,转染人HO-1基因的胰岛细胞移植后移植物存活时间为(10.56±4.33)d,两组比较,P<0.05;转染人HO-1基因的胰岛细胞培养48 h即见人HO-1蛋白表达;肝内移植7 d后移植物有人HO-1蛋白表达;移植胰岛周边及胰岛内淋巴细胞浸润程度较单纯胰岛移植组明显减轻。结论大鼠胰岛转染人HO-1基因能够增加体外培养胰岛细胞的抗凋亡能力,并能延长体内移植胰岛的存活时间,减轻淋巴细胞对胰岛的浸润。     

抗CD40L单克隆抗体在异种胰岛移植排斥反应中的作用

目的 探讨抗CD40L单克隆抗体在异种胰岛移植排斥反应中的作用及其机理.方法 建立人-大鼠异种胰岛移植模型,用抗CD40L单克隆抗体进行干预治疗,观察糖尿病大鼠行异种胰岛移植后的血糖变化,糖尿病大鼠和移植物的生存情况及移植物病理形态学改变,采用 ELISA法检测糖尿病大鼠细胞因子IL-2和TNF-α水平的变化.结果 ①全部糖尿病大鼠在胰岛移植后第(2.3±0.2) d血糖降至正常,对照组血糖在移植后第(8.1±0.6) d开始明显升高,治疗组血糖在移植后第(18.5±1.2) d开始明显升高,明显晚于对照组(P＜0.01).②治疗组移植物存活时间[(22±8.2) d]较对照组[(10±2.1) d]显著延长(P＜0.01).③糖尿病大鼠生存时间,治疗组[(35±6.5) d]显著长于对照组[(21±5.7) d ],P＜0.05.④对照组在移植后第(3.2±0.3) d IL-2及TNF-α的水平均急剧上升,至移植后第(7.3±0.5) d达到高峰; 治疗组IL-2及TNF-α的水平在移植后第(22.6±1.7) d开始明显上升,至移植后第(28.5±2.2) d达到高峰,明显晚于对照组(P＜0.01).结论 抗CD40L单克隆抗体可抑制异种胰岛移植的排斥反应,延长受体大鼠和移植物的存活时间.     

CTLA4Ig基因对大鼠胰岛移植后排斥反应的治疗作用

目的　研究CTLA4Ig基因在糖尿病大鼠体内表达及其产物对胰岛移植物存活的作用。方法　利用Lipofectin载体包裹CTLA4IgcDNA质粒后转染鼠胰岛和肌肉细胞 ,检测移植后CT LA4Ig表达和T淋巴细胞转化率。结果　胰岛移植术后 7dT淋巴细胞转化试验 ,实验组 (A组 )和对照组 (B组 )每分钟脉冲数 (cpm)分别为175 .7± 98.2 ,2 5 4.4± 116 .3 ,两组比较差异显著 (P <0 .0 5 )。A组胰岛移植第 7d ,2只大鼠血清CTLA4Ig呈阳性 (阳性率 2 0 % )。A、B两组胰岛移植后血糖维持正常时间分别为 (14.8± 12 .3)d和 (3 .6± 5 .1)d ,两组比较差异显著 (P <0 .0 5 )。A、B两组大鼠平均存活时间分别为 (2 4.0± 10 .8)d和 (10 .8± 4.8)d ,两组比较 ,差异有极显著性 (P <0 .0 1)。结论　脂质体包裹的CTLA4IgcDNA转染肌细胞和胰岛细胞 ,可以在受体大鼠胰岛细胞或肌肉组织中表达 ,其表达产物可使胰岛移植物和受体鼠存活时间明显延长 ,抑制细胞免疫活性 ,发挥其治疗排斥反应的作用     

CTLA4Ig和西罗莫司短期联合使用延长异种胰岛移植物的存活

目的 观察细胞毒性T淋巴细胞相关抗原4融合蛋白(CTLA4Ig)与西罗莫司(SRL)联用阻断共刺激通路对异种胰岛移植物存活的影响.方法 取C57BL/6小鼠,腹腔注射链佐星,制成糖尿病模型.采用随机单位组设计分组法将糖尿病小鼠分为7组,各组均于小鼠左肾包膜下移植SD大鼠胰岛300胰岛当量.CTLA4Ig组分别于移植当天及移植后第2、4、6天腹腔注射CTLA4Ig 0.5 mg/d;SRL组分别于移植当天及移植后第1、2天给予SRL灌胃,0.2 mg·kg-1·d-1,其后隔天用药1次,共用2周;MRI组分别于移植当天及移植后第2、4天腹腔注射仓鼠抗小鼠CD154单克隆抗体(MR1)0.5 mg/d;CTLA4Ig和SRL联用组(SRL联用组)、CTLA4Ig和MRl联用组(MR1联用组)以及CTLA4Ig、MR1和SRL联用组(三药联用组)各药物的剂量与用法同上述各组;对照组仅行胰岛移植,不予以药物.观察至移植后200 d,通过监测受者血糖水平来判断排斥反应的发生情况.记录各组移植物的存活(即无排斥反应)时间.发生排斥反应者,或未发生排斥反应、移植物存活时间＞200 d者,取移植胰岛,行HE染色及免疫荧光染色,进行组织学观察.结果 对照组移植物存活时间中位数为17 d,该组最终均发生排斥反应.SRL组、MRl组和CTLA4Ig组移植物存活时间中位数分别为34 d、98 d和77 d.均明显长于对照组(P＜0.05),三组中分别有90%(9/10)、62.5%(5/8)和83.3%(5/6)的小鼠发生排斥反应.SRL联用组移植物存活时间中位数为130 d,明显长于上述4组(P＜0.01),有50%(3/6)的小鼠发生排斥反应.MR1联用组以及三药联用组移植物存活时间中位数均＞200 d,分别有42.9%(3/7)和25%(2/8)的小鼠发生排斥反应.组织学检查结果显示,对照组发生排斥反应时,其移植胰岛破坏严重,可见大量CD4+和CD8+淋巴细胞及巨噬细胞浸润,并可见IgG、IgM和补体C3沉积.其它组发生排斥反应者的组织学改变与对照组相似.SRL联用组存活200 d的小鼠,其移植胰岛组织中未见或仅有少量炎症细胞浸润,胰岛素和胰高血糖素染色阳性,未见IgG、IgM和补体C3沉积.结论 短期联合使用CTLA4Ig和SRL能显著延长小鼠体内大鼠来源的胰岛的存活时间.     

Prolonged xenograft survival of islets infected with small doses of adenovirus expressing CTLA4Ig.

BACKGROUND: Systemic administration of the inhibitor of costimulation, CTLA4Ig, has been shown to prolong islet graft survival. The purpose of this study was to compare local and systemic expression of murine CTLA4Ig in transplants of rat islets into mice. METHODS: Murine CTLA4Ig was made by joining two polymerase chain reaction products, the extracellular portion of CTLA4 and the Fc portion of IgG2a. Recombinant adenovirus expressing CTLA4Ig (AdCTLA4Ig) was generated using the strategy of Cre-lox recombination. Isolated rat islets infected with AdCTLA4Ig at multiplicities of infection (MOIs) ranging from 0.1 to 10 were transplanted into streptozocin diabetic male B6AF1 mice. Control islets were mock infected or infected with AdLacZ or AdsIg, a recombinant adenovirus expressing only the Fc portion of IgG2a. Also, AdCTLA4Ig and control viruses were injected intramuscularly into mouse transplant recipients at the time of islet transplantation to provide CTLA4Ig systemically. RESULTS: Control islets transplanted into diabetic mice were rejected in 13-17 days. Islets infected with AdCTLA4Ig had dose-dependent prolongation of graft survival. Prolonged survival was even found with very low MOIs of 0.1 and 0.5, with survivals of 24+/-4.2 and 25+/-2.2 days, respectively. Survival with an MOI of 10 was 39+/-8.7 days. With intramuscular injection, no prolongation was found at the lowest relative MOIs of 0.2 and 1, but there was dose-dependent prolongation of graft survival with larger doses. At the highest relative MOI of 400, survival was prolonged to 58+/-10 days. CONCLUSIONS: Rat islets infected with AdCTLA4Ig transplanted into mice had prolonged graft survival. Prolonged survival with MOIs as low as 0.1 and 0.5 indicates that only a minority of islet cells need to express CTLA4Ig to exert an effect. Moreover, the results suggest that the improved islet graft survival is due to a local influence of CTLA4Ig.     

CTLA4Ig基因和CD40Ig基因转染供肾对异种大鼠移植肾存活的影响

目的 探讨细胞毒性T淋巴细胞相关抗原4融合蛋白(CTLA4Ig)基因和CD40Ig基因转染供肾对异种大鼠移植肾存活的影响.方法 以PcDNA3.1质粒为载体,通过脂质体2000将CTLA4Ig基因和CD40Ig基因转染豚鼠肾脏,再移植(异位肾移植)给SD大鼠.实验分4组进行:第1组供肾以PcDNA3.1空载体脂质体复合物转染(空载体组);第2组供肾转染CD40Ig基因(CD40Ig转染组);第3组供肾转染CTLA4Ig基因(CTLA4Ig转染组);第4组供肾同时转染CTLA4Ig基因和CD40Ig基因(双基因转染组).术后观察各组血清肌酐、移植肾组织病理改变以及移植肾存活时间.结果 空载体组、CD40Ig转染组、CTLA4Ig转染组和双基因转染组受者的存活时间分别为(6.8±1.9)d、(40.7±10.9)d、(49.3±9.5)d和(75.7±8.0)d,3个转染组明显长于空载体组(P＜0.01),其中双基因转染组移植肾存活时间最长,与其他3组比较,差异均有统计学意义(P＜0.01).各组术后血清肌酐水平呈上升趋势,但升高幅度以双基因转染组为最低(P＜0.01).术后第30天,CD40Ig转染组和CTLA4Ig转染组存活大鼠的移植肾组织中可见大量淋巴细胞浸润,而双基因转染组的移植肾组织中仅见少量淋巴细胞浸润.结论 供肾局部同时转染CTLA4Ig基因和CD40Ig基因可明显延长其异种移植后的存活时间.     

Effect of Triple Costimulation Blockade on Islet Allograft Survival in Sensitized Mice

Background

Islet allograft rejection in sensitized recipients is difficult to control by costimulation blockade using anti-CD154 and cytotoxic T-lymphocyte antigen-4 immunoglobulin (CTLA4Ig). Because leukocyte function antigen (LFA) 1 is highly expressed on memory T cells, adding an LFA-1 blockade may inhibit memory T-cell activities. We examined the effects on islet allograft survival of triple costimulation blockade in presensitized recipient mice.

Methods

C57BL/6 mice were sensitized by transplantation under the kidney capsule or intraperitoneal injection of Balb/c islets. Four weeks after transplantation, sensitization was confirmed by flow-cytometric detection of alloreactive antibodies. Diabetes was induced by a single intravenous injection of streptozotocin. Recipients were transplanted with 200 Balb/c islets under the right kidney capsule. Graft function was assessed by daily blood glucose and body weight records. Transplanted animals were divided into 3 treatment groups: group 1, control antibody; group 2, anti-CD154 and CTLA-4 Ig double therapy; group 3, anti-CD154, CTLA4Ig, and anti-LFA-1 triple therapy. Injections were administered every second day from day −2 to day 8.

Results

Naïve mice rejected islet allografts between days 7 and 29 (mean 16 ± 6 d; n = 5), sensitized mice in group 1 between days 0 and 14 (mean 7 ± 5 d; n = 8), in group 2 between days 4 and 16 (mean 8 ± 4 d; n = 7), and in group 3 between days 4 and 26 (mean 11 ± 7 d; n = 10).

Conclusion

Triple costimulation blockade with anti-CD154, CTLA4Ig, and anti-LFA-1 was not sufficient to improve islet allograft survival in sensitized recipients.     

Ex vivo and systemic transfer of adenovirus-mediated CTLA4Ig gene combined with a short course of FK506 therapy prolongs islet graft survival

Adenovirus-mediated CTLA4Ig gene transfer has been reported to enhance graft survival in several rodent transplantation models. In this study, we investigated the efficacy of ex vivo and systemic transfer of the CTLA4Ig gene by adenoviral vectors in pancreatic islet allo-transplantation. Islet grafts from BN rats were transplanted to chemically induced diabetic LEW rats. First, ex vivo CTLA4Ig gene transfer into isolated islets was performed prior to transplantation. Survival of transduced grafts under the kidney capsule was slightly prolonged (8.6+/-1.3 days) compared with survival of untransduced grafts (6.7+/-1.2 days); when combined with a short course of FK506, graft survival was further extended (32.6+/-10.7 days vs. 13.7+/-1.0 days with FK506 alone). Secondly, systemic gene transfer was accomplished by intravenous administration immediately after the transplantation procedure. In these animals, islet grafts under the kidney capsule survived longer (15.2+/-3.3 days) than in controls (6.7+/-1.2 days), and when FK506 was administered perioperatively, all the islet grafts survived for more than 100 days. In systemically transduced recipients, the survival of islet grafts transplanted into the liver was not significantly different from that of the grafts placed under the kidney capsule. In order to examine organ-specific immunogenicity, heterotopic BN cardiac grafts were transplanted to LEW rats intra-abdominally, with the virus transferred systemically as in the islet model. In contrast to the islet grafts, all the cardiac grafts were accepted for longer than 100 days, even without FK506 therapy. Finally, the LEW recipients with long-surviving islet or cardiac grafts were re-transplanted with islet grafts from the same donor strain (BN) on day 100. The second islet grafts survived longer than 100 days in half of the cardiac recipients, but consistently failed in the islet recipients. We conclude that in this transplant model, CTLA4Ig gene transfer and FK506 treatment synergistically improved islet graft survival, systemic transfer of the gene was more effective than ex vivo transfer to the islets, and donor-specific tolerance could not be achieved for islet transplantation but was achieved for cardiac transplantation.     

Effects of Gene Transfer CTLA4Ig and Anti-CD40L Monoclonal Antibody on Islet Xenograft Rejection in Mice

Blockade of a costimulatory pathway by adenovirus-mediated cytotoxic T lymphocyte associated antigen 4 immunoglobulin (CTLA4-Ig) gene transfer and anti-CD40L mAb(MR1) have been reported to enhance graft survival in several experimental transplantation models. In this study, we investigated the effects of gene transfer of CTLA4Ig and MR1 on islet xenograft rejection in mice. Recombinant adenovirus AdCTLA4Ig was constructed to express CTLA4Ig. Islet grafts from adult male DA rats transferred with AdCTLA4Ig were transplanted to streptozocin-induced diabetic Balb/c mice. The diabetic mice were treated with MR1 after transplantation. We evaluated the islet xenograft mean survival time as well as changes in interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) levels in transplanted mice. The mean survival of islet xenografts in the MR1 treatment group was 34.9 ± 5.62 days, in the AdCTLA4Ig treatment group it was 56.5 ± 10.64 days, and in the AdCTLA4Ig plus MR1 treatment group it was 112.9 ± 19.26 days, all significantly prolonged compared with an untreated group (8.1 ± 0.83 days). Within 1 week after transplantation the levels of IL-2 and TNF-α showed sharp increases in the untreated group, being significantly higher than those observed prior to transplantation. In conclusion, using both AdCTLA4Ig and MR1 can improve the islet xenograft survival. The beneficial effects of the combined use of the 2 reagents were superior to either 1 alone, possibly related to down-regulated expression of Th1 cell-related cytokines.     

Adenovirus-mediated CTLA4Ig or CD40Ig gene transfer delays pancreatic islet rejection in a rat-to-mouse xenotransplantation model after systemic but not local expression

Transient costimulation signal blockade of either CD28/CD80-86 interactions and/or CD40/CD154 interactions can prevent islet rejection in some models of both allo- and xenotransplantation. We have used adenoviruses coding for CTLA4Ig or CD40Ig and compared the efficacy of genetic modification of islets to systemic production through either intramuscular (i.m.) or intravenous (i.v.) injection of these vectors in a rat-to-mouse islet transplantation model. When gene transfer was performed into islets, a high level of primary nonfunction was induced. Furthermore, transduced functional grafts were rejected with the same kinetics as nontransduced islets. In contrast, i.m. AdCTLA4Ig and i.v. AdCD40Ig significantly delayed rejection (mean survival time of 54 +/- 26.9 and 67.6 +/- 44.9 days, respectively, vs. 24.3 +/- 9.7 days for unmodified islets, p < 0.05). Combination of ex vivo AdCTLA4Ig islet transduction and i.v. AdCD40Ig did not improve graft survival further. In conclusion, islet graft transduction with adenoviruses coding for costimulation inhibitors resulted in local expression with low serum concentrations of CTLA4Ig or CD40Ig and was unable to protect islet xenografts from rejection. In contrast, i.m. or i.v. gene transfer resulted in high serum concentrations of these molecules and was highly efficient in prolonging xenograft survival. These results contrast with the efficacy of AdCTLA4Ig we observed in a rat islet allotransplantation model and suggest that islet xenograft rejection might be more difficult to control.     

移植物局部转染CTLA4Ig基因对大鼠小肠移植排斥反应的预防作用

目的探讨CTLA4Ig基因在移植小肠局部转染表达及表达产物对排斥反应的预防作用。方法大鼠小肠移植前,经肠系膜上动脉给供肠灌注脂质体包裹的CTLA4IgcDNA重组质粒,术后应用组织免疫学及逆转录聚合酶链反应(RTPCR)检测移植小肠中CTLA4Ig转基因产物的表达,并行组织病理学检查,同时设立无CTLA4Ig基因转染的对照组。结果对照组(n=8)的移植小肠在术后7～14d全部坏死;实验组(n=18)7只受鼠的移植小肠于术后56～90d坏死,11只受鼠的移植小肠于术后90d时仍存活良好。对照组移植物组织切片见单核细胞广泛浸润,肠壁肌层及黏膜出血坏死,绒毛及隐窝结构消失;实验组存活超过90d者组织切片仅见少量单核细胞浸润,黏膜轻度增厚,但无明显的肠绒毛缩短及隐窝细胞减少等改变。组织免疫学及RTPCR结果显示,在移植后28d内移植小肠中有CTLA4Ig转基因产物的表达,但第56d及90d则检测不到其表达。结论经肠系膜上动脉体外灌注脂质体包裹的CTLA4IgcDNA重组质粒可有效转染移植小肠,CTLA4Ig基因在移植小肠局部转染对术后的排斥反应具有预防作用。