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.     

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     

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。     

CTLA4Ig与CD40Ig双基因局部共转染对异种大鼠移植肾Bcl-2/Bax的影响

目的:探讨CTLA4Ig与CD40Ig双基因局部转染对异种移植肾Bcl-2/Bax的影响,探讨其诱导免疫耐受的机制。方法:以PcDNA3.1(+)-CTLA4Ig和PcDNA3.1(+)-CD40Ig为载体,通过脂质体lipo2000将CTLA4Ig与CD40Ig双基因转入豚鼠肾脏,以豚鼠为供者,SD大鼠为受者,行异种肾移植手术。将豚鼠到SD大鼠的异种肾移植模型分为pcDNA3.1空载体组(第1组)、CD40Ig基因局部转染组(第2组)、CTLA4Ig基因局部转染组(第3组)和CD40Ig、CTLA4Ig双基因局部转染组(第4组)。Western blot检测移植肾HA-CTLA4Ig、HACD40Ig蛋白表达和移植肾Bcl-2、Bax的表达情况,观察移植肾病理改变。结果:术后第5天,第4组移植肾淋巴细胞浸润明显少于第2组和第3组,第4组移植肾Bcl-2蛋白表达明显升高(P0.05),而Bax蛋白表达则显著降低(P0.05)。结论:CTLA4Ig与CD40Ig双基因局部转染供肾可降低Bax表达,上调Bcl-2表达,诱导移植肾免疫耐受。     

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     

重组腺病毒载体局部介导CTLA4Ig延长小鼠异体移植皮肤的存活

目的 寻求一种简单可行的延长异体移植皮肤存活的方法。方法 将CTLA4Ig cDNA插入重组腺病毒载体柯氏质粒中,再通过同源重组构建CTLA如一重组腺病毒载体;观察该表达载体对体外培养的皮肤组织块的转染情况;并应用该载体转染皮肤移植物及局部创面,观察其对昆明小鼠→Balb c小鼠背部创面异体移植皮肤存活期的影响。结果 CTLA4Ig重组腺病毒载体能成功转染并表达于体外培养的皮肤组织块中;局部应用CTLA4Ig重组腺病毒载体使小鼠异体移植皮肤存活期延长至21d。结论用腺病毒载体局部转染CTLA4Ig能有效延长异体移植皮肤的存活。     

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     

肠移植早期黏膜地址素细胞黏附分子在大鼠移植小肠及其肠系膜淋巴结的表达

目的 探讨黏膜地址素细胞黏附分子（MAdCAM-1）在大鼠小肠移植早期移植肠及其肠系膜淋巴结中的表达及意义。方法 选用近交系F344／N和BN大鼠建立全小肠异位移植模型后分3组：第1组，非手术对照组（F344／N）；第2组，同基因移植组（F344／N→F344／N）；第3组，异基因移植组（BN→F344/N）。术后1、3、5、7d取各组移植肠及其肠系膜淋巴结检测MAdCAM-1表达的分布及变化，同期进行移植肠组织病理学检查。结果 同基因移植组各检测时点的肠黏膜组织表现与正常小肠的组织学特征基本相同；异基因移植组肠黏膜组织表现符合轻-中-重度排斥反应的渐进过程，2周后移植肠绒毛变的低平，散在黏膜上皮脱落，移植肠相关肠系膜淋巴结萎缩明显。MAdCAM-1在急性排斥反应期，高表达于移植肠固有层及其肠系膜淋巴结，特别是高表达于肠黏膜固有层中的扁平血管内皮细胞表面。同基因移植组术后MAdCAM-1的表达在1—7d均无明显量的变化；而异基因移植组MAdCAM-1在移植肠中的表达呈上升趋势，而在其肠系膜淋巴结中的表达呈下降趋势。结论 MAdCAM-1与小肠移植急性排斥反应的进展关系密切。     

大鼠供心转染CTLA4-Ig基因抑制心脏移植后排斥反应

目的研究大鼠供心转染CTLA4-Ig基因抑制心脏移植术后排斥反应的可行性。方法以BN大鼠为供者,Lewis大鼠为受者,建立心脏移植模型。将实验分为2组。对照组:供心获取过程中不给予任何干预处理;实验组:在供心获取的过程中,以质粒载体携带CTLA4-Ig(pUF1-CTLA4-Ig)经过冠状动脉灌注供心。采用逆转录聚合酶链反应(RT-PCR)检测供心组织中CTLA4-IgmRNA的表达,观察移植心存活时间;酶联免疫法(ELISA)检测血清γ-干扰素(IFN-γ)和白细胞介素-4(IL-4)水平;观察移植心的组织学改变;用免疫组织化学SABC法检测移植心组织中CD4 和CD8 T细胞的浸润。结果实验组移植心存活时间较对照组明显延长[(11.70±1.24)dvs(5.62±0.74)d,P<0.05]。移植后5d,实验组移植心组织中可见CTLA4-IgmRNA的表达;对照组病理学检查可见心肌间质出现弥漫性的炎性细胞浸润,伴有局部的心肌坏死,组织间质水肿,实验组仅有局灶性血管外周及心肌间质内炎性细胞浸润,未见坏死;对照组移植心组织中浸润的CD4 和CD8 T细胞数量明显高于实验组(P<0.01);实验组血清IFN-γ水平明显低于对照组(P<0.01),但血清IL-4水平差异无统计学意义(P>0.05)。结论经冠状动脉灌注转染CTLA4-Ig基因,可以抑制心脏移植后排斥反应。     

利用腺病毒载体导入CTLA4Ig基因对大鼠同种心脏移植免疫抑制作用的研究

目的　探索CTLA4Ig基因在实验动物体内的表达以及对大鼠同种心脏移植后的免疫抑制作用。方法　利用腺病毒作载体 ,将CTLA4Ig基因导入同种心脏移植的受体鼠体内 ,以编码 β 半乳糖苷酶的有复制缺陷的腺病毒重组体 (Adex/LacZ)为对照 ,观察该基因在实验动物体内的表达以及对同种异体心脏移植后的免疫抑制作用。逆转录多聚酶链反应 (RT PCR)法检测CTLA4Ig基因在大鼠体内的表达 ,并用流式细胞仪来测定血中CTLA4Ig蛋白质的浓度变化。 结果　导入的CT LA4Ig基因能够在大鼠肝脏中表达 ,血中CTLA4Ig的浓度明显高于对照组 (P <0 .0 5 ) ,而且移植后心脏的存活时间显著长于对照组 (P <0 .0 1)。结论　CTLA4Ig基因能在大鼠体内良好表达并明显拮抗同种移植排斥反应     

CTLA4IgG treatment induces long-term acceptance of rat small bowel allografts

BACKGROUND: CTLA4 immunoglobulin (Ig)G that binds to B7 effectively inhibits the signaling of CD28/CTLA4-B7 pathway and induces antigen specific T cell unresponsiveness in vitro and in vivo. Using CTLA4IgG, we examined induction of long-term graft survival and the mechanism of maintenance of tolerance in rat allogeneic small bowel transplantation. METHODS: Small bowels of Brown-Norway rats (RT1n) were heterotopically transplanted into Lewis rats (RT1l). Recipients were treated with an i.p. injection of either CTLA4IgG or control IgG for 7 days. RESULTS: Long-term survival was observed in rats treated with CTLA4IgG, whereas control rats died within 16 days after transplantation. To examine whether a tolerant state was established in long-term survival rats, secondary transplantation was performed using small bowels of Brown-Norway rats or ACI (RT1b) rats. It was demonstrated that small bowels of Brown-Norway rats were accepted; however, those of ACI rats were rejected within 10 days. Serum concentrations of interleukin (IL)-4 were maintained at >50 microg/ml for 7 days after transplantation in rats treated with CTLA4IgG but <15 microg/ml in control rats. IL-2 concentration was reduced to half in CTLA4IgG-treated rats compared with that in control recipients. Serum IFN-gamma in CTLA4IgG-treated recipients increased after transplantation and was not distinguishable from that of control recipients during the first 7 days after transplantation. Conclusion. We demonstrated that CTLA4IgG treatment alone for 7 days induced a long-term donor specific tolerance in rat allogeneic small bowel transplantation. The induction of long-term acceptance of small bowel allografts by CTLA4IgG is not caused by simply the shift of anti-alloimmune responses from Thl to Th2 cytokine production.     

Prevention of Acute Lung Allograft Rejection in Rat by CTLA4Ig

CTLA4 immunoglobulin (CTLA4Ig), which binds with a high affinity to B7-1 and B7-2, interrupts T-cell activation by inhibiting costimulatory signal. CTLA4Ig has been used in hopes of achieving antigen-specific tolerance induction in several solid organ transplants. In lung allograft rejection, however, its use has been controversial in terms of its effect on prevention of rejection. In the present study, the effect of murine CTLA4Ig on rat-lung allograft rejection was investigated. Rat left-lung transplantation was performed in an RT1 incompatible donor (Brown Norway; BN)-recipient (F344) combination. All allografts (n = 12) without any treatment were rejected within 7 days after transplantation. A single injection of murine form CTLA41g at a dose of 100 microg intraperitoneally (ip) or intravenously (iv) on day 1 post-transplantation achieved long-term graft survival (>90days) in 2/5 (40%) and 3/8 (38%), respectively. Moreover, 6/7 (86%) allografts in rats that received iv injection of 500 microg CTLA4Ig survived more than 90days. Allograft survival in the CTLA4Ig 500 microg iv recipient group was significantly longer than that in the no-treatment control or control immunoglobulin group (p <0.01). Four out of seven recipients bearing functional allografts for more than 90 days with the CTLA4Ig treatment accepted donor-specific skin grafts, whereas all third-party skin grafts (n=3) were rejected. Prevention of rat-lung allograft rejection could be achieved by intravenous administration of CTLA4Ig, resulting in long-term allograft survival with acceptance of donor-specific skin grafts.     

Adenovirus‐mediated gene transfer of CTLA4lg gene results in prolonged survival of heart allograft

Abstract It has been demonstrated that the administration of CTLA4Ig protein can induce the suppression of allograft and xenograft rejection. The purpose of this study is to determine the effect of adenovirus‐mediated gene transfer with CTLA4Ig gene on the transgene expression and suppression of alloimmune response in allogeneic cardiac transplantation of rats. Adenoviral vectors with β galactosidase or human CTLA4Ig cDNA (Adex/LacZ, Adex/hCTLA4Ig) were constructed. These vectors were transduced to the liver of Lewis (LEW) rats by intravenous injection. Then LEW rats received heterotopic cardiac transplantation from Dark Agouti rats. Experiments were performed using both CTLA4Ig gene transduction and/or immunosuppressant FK506. The transgene expression after adenovirus‐mediated transfer with CTLA4Ig cDNA lasted for several months, compared with several weeks after that in controls, which was proportional to the blood concentration of CTLA4Ig protein. By the administration of 1 × 10⁹ PFU of Adex/hCTLA4Ig, the survival of cardiac grafts was significantly prolonged, compared with controls or the use of 1 × 10⁸ PFU of Adex/hCTLA4Ig. In the rats with beating grafts over 100 days, the blood concentrations of CTLA4Ig were undetectable. The combination therapy using a low titer Adex/hCTLA4Ig and low‐dose of FK506 was synergistically effective on this cardiac transplantation model. In conclusion, adenovirus‐mediated gene transfer with CTLA4Ig gene was efficient for the prolongation of both transgene expression and allograft survival.     

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。     

Gene therapy for organ grafts using rapid injection of naked DNA: application to the rat liver

BACKGROUND: We developed a nonviral gene transfer method using rapid injection of naked DNA targeting the liver and applied it in a rat model of liver transplantation. METHODS: Inbred Dark Agouti and Lewis rats were used. To test the efficacy and adverse effects of systemic or local (catheter-based) injection, different volumes of phosphate-buffered saline containing naked DNA encoding beta-galactosidase (lacZ) were injected. Luciferase expression was followed by non-invasive imaging, and a cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4Ig) protein was tested functionally by allogenic heart transplantation. Gene transfer was then tested in rat auxiliary liver transplantation (ALT) and orthotopic liver transplantation (OLT). The timing of gene transfer was evaluated in the auxiliary liver transplantation model, and OLT was performed using a liver graft to which luciferase or the CTLA4Ig gene was transferred 2 days before. RESULTS: LacZ was expressed extensively in a volume-dependent manner; however, a large volume often induced recipient death. After local delivery of CTLA4Ig cDNA to the liver, survival of Dark Agouti heart grafts lengthened with increased CTLA4Ig serum levels. Liver grafts injected with naked DNA at the time of donation did not survive, but livers grafted 2 days after gene transfer survived. Successful expression of luciferase and production of CTLA4Ig were finally confirmed in the rat that underwent OLT. CONCLUSIONS: We successfully applied a nonviral hydrodynamic gene transfer method to the rat liver and showed its potential in liver grafting. The high incidence of graft failure when this procedure is performed on the day of organ donation is a potential limitation that needs to be overcome in clinical application.     

负载Ad-CTLA4Ig的未成熟树突状细胞延长大鼠移植肾存活时间的探讨

目的 探讨负载细胞毒性淋巴细胞抗原4免疫球蛋白基因重组腺病毒(Ad-CTLA4Ig)的受者未成熟树突状细胞(DC)对大鼠移植肾存活时间的影响.方法 选择雄性SD大鼠为供者,雄性Wistar大鼠为受者,建立大鼠肾移植模型.将受者随机分为4组,每组12只.制备Ad-CTLA4Ig及受者未成熟DC悬液,37℃混合孵育6 h,于移植前7 d,实验组经腹腔内注射负载Ad-CTLA4Ig的DC悬液;Ad-CTLA4Ig对照组、重组腺病毒空载体(Ad-VG)对照组和生理盐水(NS)对照组分别经腹腔内注射1 ml Ad-CTLA4Ig、Ad-VG和NS.观察各组移植肾的存活时间、组织形态学改变、受者血液中腺病毒中和抗体滴度、血清CTLA4Ig水平及混合淋巴细胞反应(MLR)的变化.结果 实验组移植肾存活时间为(94.6±9.0)d,较各对照组显著延长:[Ad-CTLA4Ig对照组为(39.6±10.6)d,Ad-VG对照组为(8.6±2.8)d,NS对照组为(8.4±2.6)d],差异均有统计学意义(P＜0.01),实验组移植肾组织损伤程度较轻,血液中腺病毒中和抗体滴度及混合淋巴细胞反应指数均较各对照组显著减少;实验组血清CTLA4Ig水平较Ad-CTFLA4Ig对照组显著升高.结论 负载Ad-CTLA4Ig的受者未成熟DC可减少腺病毒中和抗体,维持CTLA4Ig的稳定表达,从而延长大鼠移植肾的存活时间.