大鼠不同表型树突状细胞介导肝移植免疫耐受的体外实验研究

发布时间：2017-12-31 01:10

  本文关键词：大鼠不同表型树突状细胞介导肝移植免疫耐受的体外实验研究　出处：《昆明医科大学》2012年硕士论文　论文类型：学位论文

  更多相关文章： 树突状细胞 大鼠 表型 肝移植 免疫耐受

【摘要】：[目的]建立一种实用、简单、高效的从体外获取纯度相对较高且数量较多的不同成熟状态大鼠髓源性DC的培养方法。通过调控DC的成熟,得到不同表型的DC(imDC、 smDC和mDC),并得出三者介导免疫耐受的差异性,为进一步研究smDC在器官移植免疫耐受中的作用和机制提供实验依据。 [方法]体外利用rrGM-CSF和rrIL-4诱导大鼠骨髓来源单个核细胞定向分化为DC,再用TNF-α和LPS诱导不同分化状态的DC。对三种DC进行形态、细胞因子(CK)表达格局、及对异基因淋巴细胞激活能力的鉴定,分析不同表型的DC的形态、功能和介导免疫耐受的差异,为大鼠肝移植免疫耐受的实验研究提供基础。 [结果]体外培养可得到imDC、smDC和mDC三种不同成熟状态的DC：1.smDC形态体积、细胞多形性和树突特征介于imDC和mDC之间。imDC、 smDC和mDC均高表达0X62,分别为69%～89.3%、82%-89.6%和93.8%-97.7%。MHC-Ⅱ、CD80、CD86的表达分别为imDC:68.788%、53.847%、54.58%；smDC为：83.188%、73.05%、62.017%；mDC：90.005%、83.065%、73.373%。三种DC的表型表达中,mDC最高,smDC次之,imDC最低。 2. imDC、 smDC分泌IL-1β、IL-6和IL-12显著低于mDC,但smDC分泌IL-6稍低于imDC,分泌IL-12稍高于imDC,分泌IL-1β与imDC无差异；imDC、 smDC和mDC均低水平分泌IL-10。 3.混合淋巴细胞反应(MLR)中,当刺激细胞数量相同时,imDC和smDC刺激T细胞增殖的能力无差异,但mDC刺激T细胞增殖的能力是最强的,其次是阳性对照组,再次是smDC干预阳性组,最后是imDC和smDC组。同一DC作为刺激细胞,DC数量不同时,DC数量为20×103时(即R：S=1：10)对T细胞增殖的作用是最强的,其次是5×103和10×103两组(二者间无差异),再次是1×103、3×103和5×103三组(三者间无差异)。当R：S=1：10时,CCK-8检测细胞活化吸光度(absorbance, A)值：imDC(0.355±0.033)和smDC(0.412±0.018)显著低于mDC(2.283±0.222),而smDC干预阳性组(1.418±0.057)也低于mDC和阳性对照(PC)组(1.887±0.07);刺激指数(SI)：imDC组(1.18)和smDC组(1.37)明显小于mDC组(7.61),smDC干预阳性组(4.73)低于mDC组与PC组(6.29)。 4. smDC刺激T细胞后CD4+CD25+Tr细胞的比例(%)(23.97±2.13)较对照组(1.89±0.36)增加。 [结论]1smDC是形态和功能相对独立的树突状细胞业群。高水平表达OX-62和MHC-Ⅱ,中度表达CD80和CD86,低度分泌IL-1β、IL-6和IL-12等炎性细胞因子,是smDC诱导免疫耐受的基础。 2. imDC低-中度表达MHC-Ⅱ,低表达CD80和CD86,低分泌IL-1β、II.-6和IL-12等炎性因子,从而诱导效应T细胞的无能或凋亡产生免疫耐受。 3.mDC因高表达MHC-Ⅱ、CD80和CD86,高分泌IL-1β、IL-6和IL-12等炎性因子,从而介导免疫反应。
[Abstract]:Objective To establish a practical , simple and efficient method for obtaining DC ( imDC , smDC and mDC ) from different mature states with relatively high purity and higher quantity in vitro . By regulating the maturation of DC , different phenotypes of DC ( imDC , smDC and mDC ) were obtained , and the difference of these three factors mediated immune tolerance was obtained . The experimental basis was provided for further study on the role and mechanism of smDC in organ transplantation tolerance . The expression pattern of three kinds of DCs , the expression pattern of cytokines ( CK ) and the identification of the activation ability of allogeneic lymphocytes were analyzed . The differences of morphology , function and immune tolerance of DCs with different phenotypes were analyzed , which provided the basis for the experimental study of immune tolerance of liver transplantation in rats . imDC , smDC and mDC showed that imDC : 68.788 % , 53.847 % , 54.58 % ; smDC : 83.188 % , 73.05 % , 62.017 % ; mDC : 90.005 % , 83.65 % , 73.373 % . Among the three types of DC phenotype expression , mDC was the highest , smDC was the second , imDC was the lowest . 2 . imDC , smDC secreted IL - 1尾 , IL - 6 and IL - 12 significantly lower than mDC , but smDC secreted IL - 6 slightly lower than imDC , secrete IL - 12 slightly higher than imDC , secrete IL - 1尾 and imDC no difference ; imDC , smDC and mDC secrete IL - 10 at low levels . 3 . In mixed lymphocyte reaction ( MLR ) , the ability of imDC and smDC to stimulate T cell proliferation was not different when the number of stimulated cells was the same , but the ability of mDC to stimulate T cell proliferation was the strongest , followed by the positive control group . The value of activated absorbance ( absorbance , A ) was 1 脳 103 , 3 脳 103 and 5 脳 103 group ( 1.887 卤 0.07 ) . The stimulation index ( SI ) : imDC group ( 1.18 ) and smDC group ( 1.37 ) were significantly smaller than those in mDC group ( 7.61 ) , smDC interference positive group ( 4.73 ) was lower than mDC group and PC group ( 6.29 ) . 4 . The percentage ( % ) ( 23.97 卤 2.13 ) of CD4 + CD25 + Tr cells in T cells stimulated by smDC increased compared with that of the control group ( 1 . 89 卤 0.36 ) . Conclusion : The expression of OX - 62 and MHC - 鈪，

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