输注AdCTLA4-Ig转染的供体特异性骨髓间质干细胞诱导大鼠异体肢体移植特异性免疫耐受

发布时间：2018-04-03 19:15

  本文选题：肢体移植　切入点：免疫耐受　出处：《中南大学》2012年硕士论文

【摘要】：目的探讨经尾静脉输注含细胞毒性T淋巴细胞相关抗原4(CTLA4)分子胞外段与人工gG恒定区重组的融合蛋白(CTLA4-Ig)的重组腺病毒转染的骨髓间充质干细胞诱导大鼠同种异体肢体移植免疫耐受的可能性及其机理,为临床肢体移植诱导免疫耐受提供新的思路和方法。 方法(1)采用Adeasy同源重组腺病毒载体系统,构建携带融合基因CTLA4-Ig的重组腺病毒,测定滴度；并用其转染骨髓间充质干细胞,RT-PCR检测外源基因CTLA4-Ig的表达。(2)构建肢体移植大鼠模型,随机分为A、B、C、D四组。A组：空白对照组；B组：静脉注射BMSCs;C组：静脉注射AdCTLA4-Ig;D组：静脉注射AdCTLA4-Ig转染的BMSCs。检测移植肢体发生排斥反应时间、存活时间；观测移植肢体皮肤病理形态学改变,ELISA方法检测移植大鼠细胞因子工L-2的水平变化。 结果(1)成功构建了携带基因CTLA4Ig的重组腺病毒,滴度为1.0*109efu/ml,并能有效感染骨髓间质干细胞,使骨髓间质干细胞表达CTLA4Ig。(2)肢体移植物存活时间,空白对照组、输注AdCTLA4Ig、输注BMSCs、输注AdCTLA4-Ig转染的BMSCs组分别为7.20±0.45d、10.04±0.55d、10.04±0.55d、19.40±1.14d。各治疗组移植物存活时间均显著长于对照组(P0.05)；输注AdCTLA4-Ig转染的BMSCs组植物存活时间均显著长于输注BMSCs、输注AdCTLA4-Ig组；输注BMSCs、输注AdCTLA4-Ig组无明显差别。(3)肢体移植物排斥反应出现时间,空白对照组、输注AdCTLA4Ig、输注BMSCs、输注AdCTLA4-Ig转染的BMSCs组分别为4.40±0.55d、7.20±0.45d、7.20±0.45d、14.40±0.55d。各治疗组移植物存活时间均显著长于对照组(P0.05)；输注AdCTLA4-Ig转染的BMSCs组植物存活时间均显著长于输注BMSCs、输注AdCTLA4-Ig组；输注BMSCs、输注AdCTLA4-Ig组无明显差别。(4)肢体移植术后5天细胞因子工L-2水平,空白对照组、输注BMSCs、输注AdCTLA4Ig、输注AdCTLA4-Ig转染的BMSCs组分别为159.83±9.85pg/ml、119.54±13.46pg/ml、118.79±12.88pg/ml、64.32±15.24pg/m1。各治疗组移植物存活时间均显著长于对照组(P0.05)；输注AdCTLA4-Ig转染的BMSCs组植物存活时间均显著长于输注BMSCs、输注AdCTLA4-Ig组；输注BMSCs、输注AdCTLA4-Ig组无明显差别。(5)肢体移植术后5天移植肢体皮肤病理检查,空白对照组可见重度淋巴细胞浸润；输注BMSCs和输注AdCTLA4-Ig组可见中度淋巴细胞浸润；输注AdCTLA4-Ig转染的BMSCs组可见轻度淋巴细胞浸润。 结论(1)采用Adeasy同源重组腺病毒载体系统,可快速、简便获取高滴度的重组腺病毒AdCTLA4-Ig。(2)经尾静脉输注AdCTLA4-Ig转染BMSCs可诱导大鼠肢体移植免疫耐受,并优于单独输注AdCTLA4-Ig和BMSCs。
[Abstract]:Objective to investigate the induction of allogeneic allograft from rat bone marrow mesenchymal stem cells (BMSCs) by tail vein infusion of extracellular segment of cytotoxic T lymphocyte associated antigen (CTLA4) and recombinant fusion protein CTLA4-IgA of artificial G constant region (GG constant-region) in rat bone marrow mesenchymal stem cells (BMSCs).The possibility and mechanism of limb transplantation immune tolerance,To provide a new idea and method for clinical limb transplantation to induce immune tolerance.Methods 1) the recombinant adenovirus carrying the fusion gene CTLA4-Ig was constructed by Adeasy homologous recombinant adenovirus vector system, and the expression of exogenous gene CTLA4-Ig was detected by RT-PCR in bone marrow mesenchymal stem cells.They were randomly divided into four groups: group A: group B: group B: group B: group C: group C: group D: AdCTLA4-Ig transfected BMSCs.The graft limb rejection time and survival time were detected, and the skin pathomorphology of the transplanted limb was observed. Elisa method was used to detect the level of cytokine L-2 in the transplanted rats.Results 1) A recombinant adenovirus carrying gene CTLA4Ig was successfully constructed, with a titer of 1.0 ~ 109efur / ml, which could effectively infect bone marrow mesenchymal stem cells and make bone marrow mesenchymal stem cells express CTLA4Ig.f.) the survival time of limb grafts was compared with that of blank control group.The number of BMSCs transfected with AdCTLA4 IgA, BMSCs and AdCTLA4-Ig were 7.20 卤0.45 days, 10.04 卤0.55 days, 10.04 卤0.55 days and 19.40 卤1.14 days, respectively.The survival time of grafts in each treatment group was significantly longer than that in control group (P0.05A), the survival time of BMSCs group treated with AdCTLA4-Ig was significantly longer than that of BMSCs and AdCTLA4-Ig group, and the time of limb graft rejection in BMSCs group was not significantly different from that in AdCTLA4-Ig group (P < 0.05).In the blank control group, AdCTLA4 IgA, BMSCs and AdCTLA4-Ig transfected BMSCs group were 4.40 卤0.55 d ~ 7.20 卤0.45 d ~ 7.20 卤0.45 d ~ (14.40 卤0.55) d, respectively.鍚勬不鐤楃粍绉绘�嶇墿瀛樻椿鏃堕棿鍧囨樉钁楅暱浜庡�圭収缁，

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