体外转染AdCTLA4Ig调节异体复合组织移植排斥反应及其机制研究

发布时间：2018-04-25 14:07

本文选题：异体复合组织移植 + 细胞毒性T细胞抗原4免疫球蛋白　；参考：《第四军医大学》2011年博士论文

【摘要】：异体复合组织移植(CTA)对一些颜面部缺损患者是唯一有效的修复方式。然而,移植术后长期大量应用免疫抑制剂引起的毒副作用是阻碍其进一步广泛应用的主要障碍。因此,寻找能够有效降低免疫抑制剂用量的方案就成为亟待解决的问题。CTLA4是T细胞表面的共刺激分子之一,许多实验证实,由CTLA4分子胞外段和IgGFc段组成的融合蛋白CTLA4Ig能够有效抑制异体移植后排斥反应。本课题将携带CTLA4Ig基因的腺病毒通过体外灌注的方法转染至移植物,检验这一方案对抑制免疫排斥反应的有效性并对其作用机制进行研究。目的:验证不同缺血时间对皮瓣组织异体移植后排斥反应的影响,选择最佳的灌注时间。通过体外灌注的方法向复合组织灌注腺病毒介导的CTLA4Ig基因(AdCTLA4Ig),观测CTLA4Ig局部表达情况以及对异体复合组织移植后排斥反应的调节作用,并对局部表达的CTLA4Ig发挥免疫调节作用的机制进行探索。方法:1.近交系Brown Norway(BN)大鼠下腹部皮瓣移植至Lewis大鼠作为异体复合组织移植模型。切取皮瓣后,在室温下分别放置0h、1h、2h、3h后再移植至受体。室温放置的时间加上显微缝合耗时的1小时,各组缺血时间分别为1h、2h、3h、4h,记为Isc-1、Isc-2、Isc-3、Isc-4组。观测不同缺血时间后异体皮瓣的存活情况。术后取皮瓣组织,制成病理切片,分别进行HE染色和CXCR3免疫组化染色。检测排斥反应的程度和CXCR3阳性细胞的浸润情况。 2.近交系BN大鼠下腹部皮瓣移植至Lewis大鼠作为异体复合组织移植模型。实验分为5组,分别为:移植后不进行处理作为对照组;对照病毒AdEGFP灌注组;AdCTLA4Ig灌注组;雷帕霉素治疗组;AdCTLA4Ig灌注+雷帕霉素联合治疗组。通过免疫组化、RT-PCR检测各组移植物内及受体肝、肺等器官CTLA4Ig及其基因的表达情况;ELISA法检测受体血清中CTLA4Ig蛋白的含量;观测各组异体皮瓣存活情况、病理表现;Real-time PCR检测移植物内IFN-γ、IL-4等mRNA的表达变化情况;检测受体淋巴细胞对供体和第三方大鼠淋巴细胞的混合淋巴细胞反应。 3.取AdEGFP和AdCTLA4Ig灌注组受体次级淋巴器官,制成淋巴细胞单细胞悬液,进行CD4~+CD25~+Foxp3~+调节性T细胞染色,流式细胞术检测调节性T细胞的比例,比较调节性T细胞在两组之间的差异。分别取AdEGFP和AdCTLA4Ig灌注皮瓣,Real-time PCR检测皮瓣内吲哚胺-2,3双加氧酶(IDO)mRNA及皮瓣内Foxp3 mRNA的含量,比较两组之间的差异。结果:1. Isc-1、Isc-2、Isc-3、Isc-4各组移植皮瓣的存活时间分别为:9.0±1.2天、8.6±1.1天、8.8±1.3天、7.0±0.8天。Isc-4组同其他各组相比存活时间显著缩短。术后6天,Isc-4组皮瓣淋巴细胞浸润程度严重,表皮脱落,比其他各组排斥反应征象更严重。Isc-4组表达趋化因子受体CXCR3的浸润细胞比例显著高于其他各组。 2.免疫组化结果显示,术后30天灌注皮瓣中内皮细胞,血管周围组织内有CTLA4Ig蛋白的表达,而受体肝脏组织内无目的蛋白表达。RT-PCR,结果显示AdCTLA4Ig体外灌注组皮瓣在术后7、15天CTLA4Ig mRNA表达阳性,而在对照皮肤、肝、肺内的表达不明显,联合治疗组术后45天亦未见目的基因表达。Elisa法检测受体血清中CTLA4Ig的量,目的基因转染组血清中CTLA4Ig的值在25ng/ml之内。存活数据经分析表明,局部灌注AdCTAL4Ig,显著延长移植皮瓣的存活时间。联合短程、小剂量免疫抑制剂雷帕霉素后,皮瓣存活时间大大延长,最多超过60天。相比于单纯药物组,存活时间有显著差异。术后7天取移植皮瓣组织,进行PCR检测,发现目的基因灌注组免疫排斥反应的相关分子IFNγ, IL-4的mRNA表达显著降低,显示移植物局部免疫反应受到抑制。术后7天,取实验组或对照组受体脾细胞进行混合淋巴细胞反应,结果显示实验组(细胞刺激率)SI值显著低于对照组,而用第三方SD鼠脾细胞刺激时,两者无统计学差异。 3.流式细胞术检测显示,AdEGFP灌注组和AdCTLA4Ig灌注组受体淋巴细胞中调节性T细胞的含量未见变化。而AdCTLA4Ig灌注皮瓣中IDO的mRNA表达显著升高。结论:异体皮瓣常温下缺血时间超过3小时,加重异体排斥反应。所以,皮瓣灌注1小时,孵育1小时是体外灌注的合适方案。应用这种方案体外灌注腺病毒介导的CTLA4Ig基因,可获得移植物局部特异性高表达,且有效抑制异体排斥反应。体外灌注AdCTLA4Ig基因同短程、小剂量雷帕霉素有协同效应。体外灌注AdCTLA4Ig发挥免疫调节作用的机制同调高局部IDO的量有关,而与受体调节性T细胞无关。
[Abstract]:Allogeneic complex tissue transplantation ( CTA ) is the only effective way to repair some facial defects . However , it is an urgent problem to find a solution that can effectively reduce the dosage of immunosuppressive agents .

Objective : To verify the effect of different ischemic time on rejection after allotransplantation of skin flap , and to select the best perfusion time . By means of in vitro perfusion method , we observed the local expression of adenovirus - mediated 4Ig gene and the regulation of rejection after allotransplantation , and explored the mechanism of immunoregulatory action on the local expression .

Methods : 1 . The subabdominal skin flap of the inbred line Brown Norway ( BN ) was transplanted to Lewis rats as an allograft model . After the flap was cut , the rats were placed at room temperature for 0h , 1h , 2h , 3h and then transplanted to the receptor . The time after the operation of the skin flap was 1h , 2h , 3h , 4h , respectively .

2 . The subabdominal skin flap of inbred BN rats was transplanted to Lewis rats as a model for allotransplantation . The experiment was divided into five groups : control group , control virus AdEGFP perfusion group , Adon4Ig perfusion group , rapamycin treatment group , Adon4Ig perfusion + rapamycin combination therapy group .

3 . The secondary lymphoid organs of AdEGFP and Ad4Ig perfusion group were used to make lymphocyte single cell suspension . CD4 ~ + CD25 ~ + Foxp3 ~ + regulatory T cells were stained . Flow cytometry was used to detect the proportion of regulatory T cells and to compare the difference between the two groups . AdEGFP and Ad4Ig perfusion flaps were respectively taken . Real - time PCR was used to detect the contents of indoleamine - 2,3 - dioxygenase ( IDO ) mRNA and Foxp3 mRNA in the skin flap , and the difference between the two groups was compared .

Results : 1 . Isc - 1 , Isc - 2 , Isc - 3 , Isc - 4 showed that survival time was 9.0 卤 1.2 days , 8.6 卤 1.1 days , 8.8 卤 1.3 days , 7.0 卤 0.8 days . Isc - 4 group was more serious than other groups .

2 . Immunohistochemical results showed that endothelial cells and perivascular tissues were expressed in 30 - day post - operative skin flap , but the expression of non - interest protein was not found in the tissues of the receptor . The results showed that the expression of IL - 4 Ig mRNA was not obvious in the control skin , liver and lung , but the expression of the target gene was not seen in the control skin , liver and lung .

Survival data showed that AdCTAL4Ig was infused locally . The survival time of the flap was significantly prolonged . The survival time of the flap was significantly prolonged after the combined short - range and small - dose immune inhibitor rapamycin . The results showed that the expression of IFN - 纬 , IL - 4 mRNA in the experimental group was significantly lower than that in the control group .

3 . Flow cytometry showed that the content of IDO mRNA was not changed in AdEGFP perfusion group and Ad4Ig perfusion group , but the mRNA expression of IDO increased significantly in Ad4 Ig perfusion skin flap .

Conclusion : Allogeneic skin flap is suitable for perfusion in vitro at normal temperature for more than 3 hours . Therefore , it is suitable for perfusion in vitro for 1 hour and 1 hour incubation . In vitro perfusion of adenovirus - mediated 4Ig gene can achieve local high expression of the graft , and can effectively inhibit allograft rejection . In vitro , it is synergistic to the short - range and small - dose rapamycin . In vitro , the mechanism of immunoregulation is related to the amount of high local IDO , but not related to the receptor - regulatory T cell .

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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