共刺激信号阻断剂基因局部转染对大鼠移植肾存活的影响

目的　观察CTLA 4Ig基因局部转染延长移植肾存活的效能。 方法　以CTLA 4Ig基因重组腺病毒为载体 ,将CTLA 4Ig基因转入BN大鼠肾脏。以BN大鼠为供者 ,Lewis大鼠为受者 ,行同种肾移植术。用经CTLA 4Ig基因转染的供肾移植给受者为转染组 ;用未转染CTLA 4Ig基因的供肾移植给受者为对照组。观察移植肾存活时间和术后肾功能变化。结果　转染组移植肾存活 (32± 8.0 )d ,对照组移植肾存活 (8.5± 1.4 )d ,转染组存活时间明显延长 ;转染组术后血清肌酐较同期对照组明显为低。结论　CTLA 4Ig基因局部转染供肾可明显延长移植肾的存活时间。     

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     

采用经姜黄素处理的未成熟树突状细胞延长大鼠移植肾存活时间

目的 探讨姜黄素(Cur)处理的树突状细胞(DC)诱导同种T淋巴细胞低反应性的效果以及对大鼠移植肾存活时间的影响.方法 体外培养Wistar大鼠骨髓来源的DC,经Cur处理后,以流式细胞仪检测细胞CD11c、CD80、CD86及主要组织相容性复合物(MHC)Ⅱ类抗原的变化,酶联免疫吸附试验测定DC分泌白细胞介素12(IL-12)的水平,混合淋巴细胞反应(MLR)检测其刺激Lewis大鼠T淋巴细胞增殖的能力,二次MLR测定其诱导的T淋巴细胞抗原特异性低反应性.以Wistar大鼠为供者,Lewis大鼠为受者,进行肾移植.术前第7天,经尾静脉给受者输注用Cur处理的供者DC,分设不处理对照组和未成熟DC对照组(经尾静脉注射供者的未成熟DC),术后观察移植肾存活时间及组织学改变情况,第14天检测受者T淋巴细胞对供者成熟DC的反应性.结果 Cur能明显抑制DC共刺激分子CD11c、CD80、CD86及MHCⅡ类抗原的表达以及IL-12的分泌(P＜0.05).同种T淋巴细胞对经Cur处理过的DC刺激的增殖能力明显减低,且这种低反应性具有抗原特异性.对照组和未成熟DC对照组移植肾的存活时间分别为(8.6±2.1)d和(22.4±7.4)d,实验组为(31.5±6.9)d,实验组移植肾存活时间明显长于对照组和未成熟DC对照组(P＜0.05),且其移植肾组织的损伤程度最轻.实验组受者的T淋巴细胞对供者成熟DC刺激的反应性明显低于对照组(P＜0.05),而对第三方无关抗原的刺激保持较高增殖强度.结论 Cur能抑制DC成熟功能,诱导供者特异性的T淋巴细胞低反应性,移植前输注经Cur处理的未成熟DC能显著延长大鼠移植肾的存活时间.     

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.     

腺病毒介导的hCTLA4-Ig和FasL基因转移诱导大鼠同种异体肾移植长期存活的作用

目的 探讨腺病毒介导hCTLA4-Ig和FasL基因转移延长异基因大鼠肾移植物存活时间及其相关机制.方法 供、受者分别为SD系大鼠和Wistar系大鼠,建立大鼠肾移植模型.实验分为4组:对照组、空载病毒Ad-EGFP处理组、Ad-CTLA4-Ig处理组、Ad-CTLA4-Ig+Ad-FasL处理组.对照组不予处理,其他各组在供肾冷保存时,经供肾动脉灌注(1×10~9-5 ×10~9)PFU/ml Ad-EGFP、Ad-CTLA4-Ig、Ad-CTLA4-Ig+Ad-FasL.术后比较各组的存活时间、移植肾组织学、肾功能和免疫组织化学等的改变.结果 肾移植受者平均存活时间Ad-CTLA4-Ig+Ad-FasL处理组(64.67±6.41)d、Ad-CTLA4-Ig处理组(31.33 ±6.77)d与对照组(8.17±1.17)d,空载病毒Ad-EGFP处理组(8.00±1.55)d比较明显延长(P均＜0.01),并且Ad-CTLA4-Ig+Ad-FasL处理组优于Ad-CTLA4-Ig处理组(P＜0.01);对照组和空载病毒Ad-EGFP处理组术后血清肌酐浓度迅速上升,其余两组移植肾功能保持稳定.结论 Ad-CTLA4一Ig和Ad-FasL介导的基因转移能够诱导异基因肾移植物长期存活;该效应为供体特异性,与调节性T细胞和同种反应性T细胞的删除有关.     

ICOS-ICOSL和CD28-B7共刺激信号对大鼠角膜移植排斥反应调控效应的比较

目的 观察阻断可诱导性共刺激分子及其配体(ICOS-ICOSL)或CD28-B7共刺激信号对大鼠角膜移植排斥反应的影响,并比较其差异.方法 制备腺病毒载体介导的诱导性共刺激分子融合蛋白(AdICOSIg)和腺病毒介导的细胞毒T淋巴细胞抗原4融合蛋白(AdCTLA4Ig).以DA大鼠为供者,Lewis大鼠为受者,进行角膜移植:受者分别接受经Adβ-Gal、AdICOSIg和AdCTLA4Ig转染的角膜移植,分别为局部Adβ-Gal组、局部AdICOSIg组和局部AdCTLA4Ig组;受者分别接受未经处理的角膜移植,并于移植前24 h腹腔注射Adβ-Gal、移植后48 h腹腔注射AdICOSIg或移植前24 h腹腔注射AdCTLA4Ig,分别为全身Adβ-Gal组、全身AdICOSIg组和全身AdCTLA4Ig组;以接受未经处理的角膜移植者为空白对照.术后每日于手术显微镜下观察移植角膜的透明度,以判断排斥反应的发生情况.基因转染后7 d,检测受者血清ICOSIg和CTLA4Ig的浓度以及抗腺病毒抗体水平.结果 空白对照组移植角膜存活时间为(13.1±0.3)d,局部AdICOSIg组、局部AdCTLA4Ig组和全身AdCTLA4Ig组移植角膜的存活时间长于空白对照组,分别为(14.2±0.8)d(P<0.05)、(15.8±0.6)d(P<0.01)和(22.7±4.3)d(P<0.01),局部AdCTLA4Ig组和全身AdCTLA4Ig组各有1例未发生排斥反应.全身AdCTLA4Ig组受者的血清CTLA4Ig浓度较高,其抗腺病毒载体抗体水平则较低.结论 CD28-B7与ICOS-ICOSL共刺激信号在免疫调节效应上存在差异,阻断CD28-B7信号所获得的延长移植角膜存活时间的效果优于阻断ICOS-ICOSL者.     

环孢素对共刺激阻断剂延长移植肾存活效应的影响

目的:探讨环孢素(CsA)对共刺激阻断剂CTLA4Ig延长移植肾存活效应的影响。方法:肾移植大鼠分为对照组(第1组)、CTLA4Ig组(第2组)、CsA CTLA4Ig组(第3组)、CTLA4Ig IL-2组(第4组)和CsA CTLA4Ig IL-2组(第5组),观察术后血肌酐(Scr)、移植肾病理改变、移植肾存活时间。结果:与第1组、第4组相比,第2组、第3组、第5组移植肾存活时间显著延长(P<0.01),其中,第3组移植肾存活时间最长(66.1±10.6)d;术后15天,第2组Scr显著低于第3组、第5组(P<0.05);术后30天,第3组、第5组Scr显著低于第2组(P<0.01);术后30天,第3组、第5组移植肾淋巴细胞浸润明显少于第2组。结论:CsA可增强CTLA4Ig延长移植肾存活的效应,对外源性IL-2逆转CTLA4Ig的效应具有抵抗作用。     

睾丸支持细胞与共刺激通路阻断剂对异体移植肾细胞的保护作用

目的　研究表达Fas配体 (FasL)的睾丸支持细胞与共刺激通路阻断剂细胞毒性T细胞相关抗原 4免疫球蛋白 (CTLA 4Ig)对异体移植肾细胞的保护作用 ,以提高移植肾的免疫耐受性。方法　酶消化法制备睾丸支持细胞及肾细胞。取 10 6个细胞混合移植于大鼠肾包膜下 ,实验大鼠分为 3组 :对照组 (10只 )、混合细胞移植组 (混合移植组 ,16只 )、联合CTLA 4Ig组 (CTLA组 ,10只 )。分别于术后 1、7、14、2 0d检测血清白细胞介素 2 (IL 2 )水平变化。术后 2 0d取出移植物 ,以亲和素 生物素 过氧化物酶复合物技术观察肾细胞存活状况 ;MD 2 0图像分析系统测定混合移植物灰度值 ;末端脱氧核糖核酸转移酶介导的X dUTP缺口末端标记 (TUNEL)法观察移植物中凋亡的细胞。　结果对照组无移植物存活 ;混合移植组 14只移植物存活 ,灰度值为 0 36 2± 0 0 17;CTLA组 10只移植物均存活 ,灰度值为 0 4 4 5± 0 0 2 1;混合移植组与CTLA组间灰度值差异有显著意义。术后CTLA组血清IL 2水平较混合移植组低 ,差异有显著意义 ;混合移植物中可见凋亡的淋巴细胞。　结论　异体肾细胞移植中 ,睾丸支持细胞与CTLA 4Ig对移植肾细胞具有协同保护作用。     

负载供肾抗原的致耐受性树突状细胞延长大鼠移植肾存活时间

目的探讨负载供肾抗原的致耐受性受者树突状细胞(DCs)对移植肾存活时间的影响。方法以BN大鼠为供者,Lewis大鼠为受者,Wistar大鼠为无关供者。将受者的骨髓分离培养,未经其它处理得到的DCs,将其作为DCs1;DCs1经CTLA-4Ig基因重组的复制缺陷型腺病毒(AdvCTLA-4Ig)转染后作为DCs2;DCs1先经BN大鼠供肾抗原负载后再进行AdvCTLA-4Ig转染,作为DCs3。将受者随机分为DCs1、DCs2、DCs3及对照组,每组6只。术前24h,前3组分别注入1×106个DCs1、DCs2和DCs3,对照组不注射DCs。肾移植后观察各组移植肾存活时间,术后第20d检测受者脾细胞对负载供肾抗原及无关供者肾脏抗原DCs的反应。结果DCs1、DCs2,DCs3和对照组的移植肾存活时间分别为(8.1±0.8)d、(8.8±0.9)d、(62.1±13.7)d和(8.6±0.9)d。DCs1、DCs2组与对照组比较,差异无统计学意义(P>0.05),DCs3组与对照组比较,移植肾存活时间显著延长(P<0.01)。肾移植术后,DCs3组脾细胞对负载供肾抗原的DCs反应明显低于对照组(P<0.01),而对负载无关供者抗原的DCs反应与对照组比较,差异无统计学意义(P>0.05)。结论负载供肾抗原的受者DCs经CTLA-4Ig基因修饰后具有致耐受性,可以抑制受者对供者抗原的免疫反应,明显延长移植肾的存活时间。     

重组腺相关病毒载体介导CTLA4Ig转染延长同种大鼠心脏移植存活时间

目的　研究重组腺相关病毒载体介导 CTLA4Ig(rAAV CTLA4Ig)全身转染对大鼠同种心脏移植的影响。方法　以BN大鼠为供者,Lewis大鼠为受者,建立心脏移植模型。实验分为两组。对照组:供、受者不给予任何处理;转染组:受者于心脏移植前30 d,通过尾静脉全身注射 1×109斑点形成单位(PFU)的 rAAV CTLA4Ig。观察移植心存活时间;ELISA法检测血清 CTLA4Ig、干扰素 γ(IFN γ)和白细胞介素 4(IL 4)水平;免疫组织化学法(SABC法)检测移植心组织中 CD4 和CD8 T细胞的浸润。对移植心存活时间明显延长的受者,用其脾细胞与供者脾细胞进行混合淋巴细胞培养,观察受者对供者的免疫反应状态。结果　转染组移植心存活时间较对照组明显延长(P<0.05);转染组血清CTLA4Ig蛋白一直维持在26.67~35.47 mg/L,移植后转染组血清 IFN γ水平下调,血清 IL 4水平上调(P<0.05);对照组出现典型的急性排斥反应表现,转染组心肌组织基本正常,间质内无炎性细胞浸润或血管外周及心肌间质内有局灶性炎性细胞浸润,未见坏死;对照组移植心组织中浸润的CD4 和CD8 T细胞数量明显高于转染组(P<0.01);转染组受者对供者的脾细胞增殖反应明显低于对照组(P<0.05)。结论　重组腺相关病毒载体可以介导 CTLA4Ig基因的持续表达,通过全身途径转染受者可以明显延长     

Induction of immunologic tolerance to cardiac allograft by simultaneous blockade of inducible co-stimulator and cytotoxic T-lymphocyte antigen 4 pathway

BACKGROUND: Inducible co-stimulator (ICOS) is one of the most recently described members of the CD28 family, and it plays an important role in immune responses. To investigate the role of ICOS in allograft rejection, the authors studied graft survival after cardiac transplantation in mice. METHODS: Hearts from BALB/c mice were transplanted into C3H/He mice. Immunohistochemical staining and flow cytometry were performed. Monoclonal antibody to ICOS or ICOS-immunoglobulin (Ig) was injected intraperitoneally. The authors performed mixed lymphocyte reaction (MLR). RESULTS: ICOS was expressed strongly by graft-infiltrating cells during rejection of the allograft. Blockade of the ICOS pathway with anti-ICOS antibody and ICOSIg significantly prolonged graft survival time relative to that in untreated mice; however, all cardiac allografts were eventually rejected by a single treatment. Treatment with both ICOSIg and cytotoxic T-lymphocyte antigen 4 (CTLA4) Ig induced not only long-term acceptance of the cardiac allograft but also donor-specific tolerance, which was shown by acceptance of donor but not third-party skin. Graft arterial intimal hyperplasia in these cardiac allografts was remarkably less than that in cardiac allografts treated with tacrolimus. Addition of anti-ICOS antibody or ICOSIg to MLR resulted in inhibition of T-cell proliferation. CONCLUSIONS: Inhibition of T-cell proliferation with ICOSIg and CTLA4Ig was more effective than that with ICOSIg alone. Thus, ICOS appears to be an important regulator of T-cell activation, and may be an effective therapy in clinical cardiac transplantation.     

CTLA4Ig introduced by adenovirus vector locally to prolong the survival of xenogeneic skin grafts on rat burn wounds

BACKGROUND: The purpose of this study was to explore an applicable approach for prolonging the survival of heterogenetic skin grafts on burn wounds with CTLA4Ig. METHODS: An adenovirus vector named Ad-CTLA4Ig, which could express human CTLA4Ig fusion protein, was constructed. Infecting and replicating in 293 cells, more Ad-CTLA4Ig and recombinant human CTLA4Ig (rhCTLA4Ig) were prepared, respectively. In a rat flame thermal injury model, the effect of rhCTLA4Ig on survival time of human skin graft on the eschar-excised rat burn wound was observed. Meanwhile, the efficiency of Ad-CTLA4Ig infecting cultured skin fibroblasts, keratinocytes, and partial-thickness skin samples were checked by CTLA4Ig expression essay. Then, the Ad-CTLA4Ig was administered locally on the eschar-excised wound and dermis of the skin graft, and the survival time of the human skin graft on burn wound was measured. The influence of the systemic immune function by rhCTLA4Ig and Ad-CTLA4Ig were also determined. RESULTS: The prepared rhCTLA4Ig from the supernatant of Ad-CTLA4Ig-infected 293 cells was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis sodium dodecyl sulfate poly-acrylamide gel electrophoresis and Western blot. It was found that CTLA4Ig could significantly prolong the xenogeneic skin graft survival in a dosage-dependent manner. Interestingly, the survival time was longer when CTLA4Ig was used 24 hours posttransplantation than that at hour 0. The expression of CTLA4Ig could be observed in the cultured skin fibroblasts, keratinocytes, and skin pieces soon after Ad-CTLA4Ig transfection, as demonstrated by either immunocellular chemistry or immunohistochemistry assay. When Ad-CTLA4Ig was locally administered during skin transplantation on burn wound, the survival time was increased from 7.9 days of control group to 21.6 days, whereas the systemic immune function was not affected. CONCLUSION: Administration of Ad-CTLA4Ig locally could prolong the survival time of xenogeneic skin graft on burn wound without significantly influencing the systemic immune function.     

CTLA4Ig combined with anti-LFA-1 prolongs cardiac allograft survival indefinitely

CTLA4Ig and anti-LFA-1 are members of a new generation of immunomodulatory drugs which inhibit important signaling pathways in T cell activation. Both substances target molecules which have pivitol functions in the activation of CD4+ and CD8+ T cells and have been theorized to have an interdependent relationship. These drugs have been used independently in various treatment regimens and have shown great promise in prolonging the survival of allografts. In order to test whether these substances have synergistic or potentiating effects when combined, we performed mixed lymphocyte reactions, skin transplantation and vascularised heterotopic heart transplantation in the Balb/c (H-2(d)) to C3H/HeJ (H-2(k)) strain combination. When anti-LFA-1 and CTLA4Ig were combined at low doses, there was a substantial inhibition of lymphocyte proliferation. When each drug was used as a mono-therapy in skin graft recipients, there was no significant effect on median graft survival (anti-LFA-1, 15 days; CTLA4Ig, 16 days) when compared to untreated controls (13 days), whereas a combination of anti-LFA-1 and CTLA4Ig extended graft survival significantly to 32 days. Untreated vascularised heart grafts rejected at a median of 8 days, CTLA4Ig-treated mice rejected at a median time of 79 days and anti-LFA-1-treated mice rejected at 43 days (n = 9). When CTLA4Ig and anti-LFA-1 were combined, all animals had functioning heart grafts at 100 days after transplantation. Histological analysis of combined-therapy hearts showed no signs or only minor changes associated with chronic rejection. In conclusion, these results indicate a synergistic effect of combining anti-LFA-1 with CTLA4Ig in inhibiting lymphocyte proliferation and prolonging the survival of fully MHC-mismatched allografts.     

Prolongation of renal allograft survival in rats by replication-defective recombinant adenovirus-mediated coexpression of CD40L and CTLA4Ig

BACKGROUND: Blockade of costimulatory signals has been shown to prolong allograft survival. The aim of the present study was to investigate the effect of simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways by replication-defective adenovirus-mediated expression of secretable extracellular domain of human CD40L (shCD40L) and CTLA4Ig to prolong rats renal allograft survival. METHODS: We constructed Adv-shCD40L-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig. Coexpression of shCD40L and CTLA4Ig was evaluated by confocal laser scanning microscopy. The function of these two molecules was examined in human mixed lymphocyte reactions (MLRs) in vitro and in experimental BN-to-LEWIS rat renal transplantation in vivo. RESULTS: Successful construction of Adv-shCD40L-IRES2-CTLA4Ig was confirmed by polymerase chain reaction. Coexpression of shCD40L and CTLA4Ig on human kidney cell line HK-2 cells after transfection was detected by direct immunofluorescence staining. Human MLR was inhibited to 52.2%+/-0.6% and 42.1%+/-0.2% of the vehicle control by Adv-shCD40L and Adv-CTLA4Ig, respectively. Adv-shCD40L-IRES2-CTLA4Ig resulted in further inhibition of MLR to 22.0%+/-0.2% of vehicle control. Transfection with Adv-shCD40L or Adv-CTLA4Ig alone prolonged renal graft survival to 24.8+/-2.5 days and 27.3+/-3.6 days, respectively, as compared to vehicle-treated controls (7.8+/-0.3 days). Cotransfection of both genes extended graft survival to 41.8+/-3.7 days. CONCLUSIONS: Adv-shCD40L-IRES2-CTLA4Ig, a replication-defective adenovirus carrying genes encoding human CD40L and CTLA4Ig, achieved simultaneous blockade of CD40/CD40L and CD28/B7 costimulatory pathways, Adv-shCD40L-IRES2-CTLA4 by Ig synergistically inhibited human T-cell proliferation in MLR, and prolonged rats renal allograft survival.