hIL-10基因修饰树突状细胞诱导大鼠肝移植免疫耐受的实验研究

发布时间：2019-04-08 14:15

【摘要】： 目的建立体外培养、诱导和扩增DA(Dark Agouti)大鼠骨髓来源的树突状细胞的方法;建立改良的大鼠原位肝移植模型和急性排斥反应模型,用hIL-10基因修饰大鼠骨髓来源的不成熟树突状细胞(imDC);探讨其诱导同种异体大鼠原位肝移植免疫耐受的情况。方法 1.培养、诱导和扩增DA大鼠骨髓来源的DC,经形态学、免疫表型及功能鉴定后,将pIRES2-EGFP- hIL-10质粒转染imDC,检测hIL-10在imDC中的表达。 2.建立改良的大鼠原位肝移植模型和DA大鼠→Lewis大鼠急性排斥反应模型:在原二袖套法的基础上,采用快速切取供肝,一针法连续缝合肝上下腔静脉以及对出血点进行热止血等方法,进行120例大鼠原位肝移植,并观察术后存活率。 3.行以DA大鼠为供体、近交系Lewis大鼠为受体的同种异体原位肝移植100例,随机均分为5组:未注射DC组(对照组)、mDC组、imDC组、空载体组、及hIL-10转染组;分别于移植前5d每天给Lewis大鼠腹腔注射mDC、imDC、空载体转染的imDC和hIL-10修饰的imDC各2×106个,于肝移植术后3d、7d、10d各处死4只,观察外周血肝功能变化(ALT和TBIL),肝脏病理改变,外周血清细胞因子情况,各组除去技术死亡的受鼠,将余下受鼠作生存分析,死亡时取肝脏行病理组织学检查。结果 1.成功建立了体外大量培养、诱导及扩增大鼠骨髓来源DC的方法;经倒置相差显微镜、透射电镜、扫描电镜及流式细胞仪鉴定,证实所培养细胞为DC。形态学结果显示:DC形态不规则,细胞核大而偏位,细胞表面可见细长树枝状突起。免疫表型结果显示:mDC和imDC表面均表达表面分子OX62,而共刺激分子CD86在mDC呈高表达,在imDC呈低表达。混合淋巴细胞反应(MLR)结果显示:imDC刺激同种异体T淋巴细胞的能力明显弱于mDC(P=0.005)。pIRES2-EGFP- hIL-10质粒经基因测序显示其序列与Pubmed基因库的hIL-10序列相符合;经PCR鉴定示在540bp处有明显的条带。荧光显微镜观察显示pIRES2-EGFP- hIL-10质粒转染imDC后可见黄绿色荧光。流式细胞术显示转染后DC表面分子CD86和CD80的表达未受影响。转染后MLR显示,hIL-10转染组的imDC对T细胞的增殖反应均弱于imDC组和空载体组(P=0.024和P=0.033)。ELISA检测IL-10组的hIL-10表达明显高于mDC组、imDC组及空载体组(P=0.004、0.004及0.003);经Westen-Blot检测有hIL-10蛋白表达。2.成功建立改良的大鼠原位肝移植模型和急性排斥反应模型。3.活体观察:移植后的肝脏病理组织学切片显示IL-10组术后仅有较少量的单核细胞浸润,RAI评分2～5分,属非确定型急性排斥反应到轻度急性排斥反应,10d开始出现肝细胞再生,有少量的中性粒细胞浸润。肝功能示IL-10组在7d、10d时,肝功能有所恢复,但与imDC组及空载体组相比差异无统计学意义。ELISA检测示,术后7d,IL-10组的血清IL-12浓度均低于对照组、mDC组、imDC组及空载体组(P为0.000、0.000、0.005及0.008)。IL-10组中位生存期40d均长于imDC组(23d)和空载体组(25d)(P均为0.002)。结论 1.本实验通过运用大鼠淋巴细胞分离液和培养过程的手法筛选相结合,能培养、诱导、扩增大量的DC;hIL-10基因转染imDC能够获得有效的表达,转染后对imDC表型没有明显影响;hIL-10基因转染可明显降低imDC对同种异体T淋巴细胞的反应性。 2.经改良的大鼠原位肝移植方法,手术时间短,成功率高,稳定可靠,建立了比较稳定的大鼠原位肝脏移植模型,可满足本实验的要求。 3.hIL-10基因修饰的imDC能诱导同种异体大鼠肝移植免疫耐受,显著延长同种异体肝移植大鼠的生存期。
[Abstract]:Purpose Establishment of a method of in vitro culture, induction and amplification of dendritic cells from a rat bone marrow source of a DA (Dark Agouti); an improved rat orthotopic liver transplantation model and an acute rejection response model, immature dendritic cells derived from bone marrow derived from the hIL-10 gene modified rat (imD C). To explore the immune tolerance of orthotopic liver transplantation induced by allogenic rats. case Methods 1. To culture, induce and amplify the DC of the bone marrow of the DA rats, the pIRES2-EGFP-hIL-10 plasmid was transfected imDC, and the hIL-10 was detected by transfecting the pIRES2-EGFP-hIL-10 plasmid after the morphological, immunological and functional identification. 2. Establishment of an improved rat orthotopic liver transplantation model and a model of acute rejection in the rat Lewis rats of DA rats: on the basis of the original two-cuff method, the liver and the upper and lower vena cava and the pair of upper and lower vena cava are continuously sewn by a needle method. The in-situ liver of 120 rats was performed by means of thermal hemostasis and the like. After transplantation, the survival rate was observed.3. In the case of donor and inbred Lewis rats,100 cases of orthotopic liver transplantation were randomly divided into 5 groups: no DC group (control group), mDC group, imDC group, empty vector group and hIL-10 transfection group. Lewis rats were given to Lewis on 5 days before transplantation. The imDC and hIL-10 modified imDC and hIL-10 modified imDC were injected into the rats for 3 days,7 days and 10 days after the liver transplantation. The changes of liver function in peripheral blood (ALT and TBIL), the pathological changes of the liver and the cytokines of peripheral blood were observed. In the case, the rats in each group were killed and the rest of the mice were analyzed for survival. death The results were as follows:1. A large number of methods for culturing, inducing and amplifying the bone marrow derived DC in the rat were successfully established. The results showed that the DC morphology of the cultured cells was irregular and fine. The results of the immunophenotyping show that both mDC and imDC surface express surface molecule OX62, while the co-stimulatory molecule CD The results of the mixed lymphocyte reaction (MLR) showed that the ability of imDC to stimulate the allogeneic T-lymphocytes was significantly weaker than that of mDC (P = 0.005). The pIRES2-EGFP-hIL-10 plasmid was sequenced by gene sequencing to be consistent with the hIL-10 sequence of the Pubmed gene bank. The pIRES2-EGFP-hI was observed by fluorescence microscope. L-10 plasmid was transfected with imDC, and then the yellow green fluorescence was observed. The expression of CD86 and CD80 was not affected by the expression of CD86 and CD80. The expression of hIL-10 in the IL-10 group was significantly higher than that of the mDC group, the imDC group and the empty vector group (P = 0.004, 0.004 and 0.003). -Blot detected hIL-10 protein expression. 3. In-situ liver transplantation model and acute rejection reaction model of rats.3. In-life observation: the pathological sections of the liver after transplantation show only a small amount of mononuclear cell infiltration after the transplantation of the IL-10 group, and the RAI score is 2-5, which belongs to the non-deterministic acute rejection reaction to the mild acute rejection reaction, and 10d is on. The liver function of the IL-10 group was recovered at the time of 7 d and 10 days, but compared with that of the liver function. The serum IL-12 concentration in the IL-10 group was lower than that in the control group, the mDC group, the imDC group and the empty vector group (P = 0.00), compared with the control group, the mDC group, the imDC group and the empty vector group. 0, 0.000, 0.005 and 0.008). The median survival time of the IL-10 group was longer than that of the imDC group (23d). and empty Conclusion:1. In this experiment, a large number of DC, hIL-10 and imDC were transfected by using the combination of the method of separation and culture of the lymphocytes of the rat. can obtain effective expression, and has no obvious effect on imDC phenotype after transfection; hIL-10 group 2. The modified rat orthotopic liver transplantation method has the advantages of short operation time, high success rate and stable and reliable operation. And a stable rat orthotopic liver transplantation model is established to meet the requirements of the experiment.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.4

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