本文選題：肢體移植　切入點(diǎn)：免疫耐受　出處：《中南大學(xué)》2012年碩士論文

【摘要】：目的探討經(jīng)尾靜脈輸注含細(xì)胞毒性T淋巴細(xì)胞相關(guān)抗原4(CTLA4)分子胞外段與人工gG恒定區(qū)重組的融合蛋白(CTLA4-Ig)的重組腺病毒轉(zhuǎn)染的骨髓間充質(zhì)干細(xì)胞誘導(dǎo)大鼠同種異體肢體移植免疫耐受的可能性及其機(jī)理,為臨床肢體移植誘導(dǎo)免疫耐受提供新的思路和方法。 方法(1)采用Adeasy同源重組腺病毒載體系統(tǒng),構(gòu)建攜帶融合基因CTLA4-Ig的重組腺病毒,測(cè)定滴度；并用其轉(zhuǎn)染骨髓間充質(zhì)干細(xì)胞,RT-PCR檢測(cè)外源基因CTLA4-Ig的表達(dá)。(2)構(gòu)建肢體移植大鼠模型,隨機(jī)分為A、B、C、D四組。A組：空白對(duì)照組；B組：靜脈注射BMSCs;C組：靜脈注射AdCTLA4-Ig;D組：靜脈注射AdCTLA4-Ig轉(zhuǎn)染的BMSCs。檢測(cè)移植肢體發(fā)生排斥反應(yīng)時(shí)間、存活時(shí)間；觀測(cè)移植肢體皮膚病理形態(tài)學(xué)改變,ELISA方法檢測(cè)移植大鼠細(xì)胞因子工L-2的水平變化。 結(jié)果(1)成功構(gòu)建了攜帶基因CTLA4Ig的重組腺病毒,滴度為1.0*109efu/ml,并能有效感染骨髓間質(zhì)干細(xì)胞,使骨髓間質(zhì)干細(xì)胞表達(dá)CTLA4Ig。(2)肢體移植物存活時(shí)間,空白對(duì)照組、輸注AdCTLA4Ig、輸注BMSCs、輸注AdCTLA4-Ig轉(zhuǎn)染的BMSCs組分別為7.20±0.45d、10.04±0.55d、10.04±0.55d、19.40±1.14d。各治療組移植物存活時(shí)間均顯著長(zhǎng)于對(duì)照組(P0.05)；輸注AdCTLA4-Ig轉(zhuǎn)染的BMSCs組植物存活時(shí)間均顯著長(zhǎng)于輸注BMSCs、輸注AdCTLA4-Ig組；輸注BMSCs、輸注AdCTLA4-Ig組無(wú)明顯差別。(3)肢體移植物排斥反應(yīng)出現(xiàn)時(shí)間,空白對(duì)照組、輸注AdCTLA4Ig、輸注BMSCs、輸注AdCTLA4-Ig轉(zhuǎn)染的BMSCs組分別為4.40±0.55d、7.20±0.45d、7.20±0.45d、14.40±0.55d。各治療組移植物存活時(shí)間均顯著長(zhǎng)于對(duì)照組(P0.05)；輸注AdCTLA4-Ig轉(zhuǎn)染的BMSCs組植物存活時(shí)間均顯著長(zhǎng)于輸注BMSCs、輸注AdCTLA4-Ig組；輸注BMSCs、輸注AdCTLA4-Ig組無(wú)明顯差別。(4)肢體移植術(shù)后5天細(xì)胞因子工L-2水平,空白對(duì)照組、輸注BMSCs、輸注AdCTLA4Ig、輸注AdCTLA4-Ig轉(zhuǎn)染的BMSCs組分別為159.83±9.85pg/ml、119.54±13.46pg/ml、118.79±12.88pg/ml、64.32±15.24pg/m1。各治療組移植物存活時(shí)間均顯著長(zhǎng)于對(duì)照組(P0.05)；輸注AdCTLA4-Ig轉(zhuǎn)染的BMSCs組植物存活時(shí)間均顯著長(zhǎng)于輸注BMSCs、輸注AdCTLA4-Ig組；輸注BMSCs、輸注AdCTLA4-Ig組無(wú)明顯差別。(5)肢體移植術(shù)后5天移植肢體皮膚病理檢查,空白對(duì)照組可見(jiàn)重度淋巴細(xì)胞浸潤(rùn)；輸注BMSCs和輸注AdCTLA4-Ig組可見(jiàn)中度淋巴細(xì)胞浸潤(rùn)；輸注AdCTLA4-Ig轉(zhuǎn)染的BMSCs組可見(jiàn)輕度淋巴細(xì)胞浸潤(rùn)。 結(jié)論(1)采用Adeasy同源重組腺病毒載體系統(tǒng),可快速、簡(jiǎn)便獲取高滴度的重組腺病毒AdCTLA4-Ig。(2)經(jīng)尾靜脈輸注AdCTLA4-Ig轉(zhuǎn)染BMSCs可誘導(dǎo)大鼠肢體移植免疫耐受,并優(yōu)于單獨(dú)輸注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 鹵0.45 days, 10.04 鹵0.55 days, 10.04 鹵0.55 days and 19.40 鹵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 鹵0.55 d ~ 7.20 鹵0.45 d ~ 7.20 鹵0.45 d ~ (14.40 鹵0.55) d, respectively.鍚勬不鐤楃粍縐繪鐗╁瓨?gòu)z繪椂闂村潎鏄捐憲闀夸簬瀵圭収緇，

本文編號(hào)：1706548