本文關(guān)鍵詞：大鼠不同表型樹突狀細(xì)胞介導(dǎo)肝移植免疫耐受的體外實(shí)驗(yàn)研究　出處：《昆明醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 樹突狀細(xì)胞 大鼠 表型 肝移植 免疫耐受

【摘要】：[目的]建立一種實(shí)用、簡單、高效的從體外獲取純度相對(duì)較高且數(shù)量較多的不同成熟狀態(tài)大鼠髓源性DC的培養(yǎng)方法。通過調(diào)控DC的成熟,得到不同表型的DC(imDC、 smDC和mDC),并得出三者介導(dǎo)免疫耐受的差異性,為進(jìn)一步研究smDC在器官移植免疫耐受中的作用和機(jī)制提供實(shí)驗(yàn)依據(jù)。 [方法]體外利用rrGM-CSF和rrIL-4誘導(dǎo)大鼠骨髓來源單個(gè)核細(xì)胞定向分化為DC,再用TNF-α和LPS誘導(dǎo)不同分化狀態(tài)的DC。對(duì)三種DC進(jìn)行形態(tài)、細(xì)胞因子(CK)表達(dá)格局、及對(duì)異基因淋巴細(xì)胞激活能力的鑒定,分析不同表型的DC的形態(tài)、功能和介導(dǎo)免疫耐受的差異,為大鼠肝移植免疫耐受的實(shí)驗(yàn)研究提供基礎(chǔ)。 [結(jié)果]體外培養(yǎng)可得到imDC、smDC和mDC三種不同成熟狀態(tài)的DC：1.smDC形態(tài)體積、細(xì)胞多形性和樹突特征介于imDC和mDC之間。imDC、 smDC和mDC均高表達(dá)0X62,分別為69%～89.3%、82%-89.6%和93.8%-97.7%。MHC-Ⅱ、CD80、CD86的表達(dá)分別為imDC:68.788%、53.847%、54.58%；smDC為：83.188%、73.05%、62.017%；mDC：90.005%、83.065%、73.373%。三種DC的表型表達(dá)中,mDC最高,smDC次之,imDC最低。 2. imDC、 smDC分泌IL-1β、IL-6和IL-12顯著低于mDC,但smDC分泌IL-6稍低于imDC,分泌IL-12稍高于imDC,分泌IL-1β與imDC無差異；imDC、 smDC和mDC均低水平分泌IL-10。 3.混合淋巴細(xì)胞反應(yīng)(MLR)中,當(dāng)刺激細(xì)胞數(shù)量相同時(shí),imDC和smDC刺激T細(xì)胞增殖的能力無差異,但mDC刺激T細(xì)胞增殖的能力是最強(qiáng)的,其次是陽性對(duì)照組,再次是smDC干預(yù)陽性組,最后是imDC和smDC組。同一DC作為刺激細(xì)胞,DC數(shù)量不同時(shí),DC數(shù)量為20×103時(shí)(即R：S=1：10)對(duì)T細(xì)胞增殖的作用是最強(qiáng)的,其次是5×103和10×103兩組(二者間無差異),再次是1×103、3×103和5×103三組(三者間無差異)。當(dāng)R：S=1：10時(shí),CCK-8檢測(cè)細(xì)胞活化吸光度(absorbance, A)值：imDC(0.355±0.033)和smDC(0.412±0.018)顯著低于mDC(2.283±0.222),而smDC干預(yù)陽性組(1.418±0.057)也低于mDC和陽性對(duì)照(PC)組(1.887±0.07);刺激指數(shù)(SI)：imDC組(1.18)和smDC組(1.37)明顯小于mDC組(7.61),smDC干預(yù)陽性組(4.73)低于mDC組與PC組(6.29)。 4. smDC刺激T細(xì)胞后CD4+CD25+Tr細(xì)胞的比例(%)(23.97±2.13)較對(duì)照組(1.89±0.36)增加。 [結(jié)論]1smDC是形態(tài)和功能相對(duì)獨(dú)立的樹突狀細(xì)胞業(yè)群。高水平表達(dá)OX-62和MHC-Ⅱ,中度表達(dá)CD80和CD86,低度分泌IL-1β、IL-6和IL-12等炎性細(xì)胞因子,是smDC誘導(dǎo)免疫耐受的基礎(chǔ)。 2. imDC低-中度表達(dá)MHC-Ⅱ,低表達(dá)CD80和CD86,低分泌IL-1β、II.-6和IL-12等炎性因子,從而誘導(dǎo)效應(yīng)T細(xì)胞的無能或凋亡產(chǎn)生免疫耐受。 3.mDC因高表達(dá)MHC-Ⅱ、CD80和CD86,高分泌IL-1β、IL-6和IL-12等炎性因子,從而介導(dǎo)免疫反應(yīng)。
[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 鹵 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 鹵 2.13 ) of CD4 + CD25 + Tr cells in T cells stimulated by smDC increased compared with that of the control group ( 1 . 89 鹵 0.36 ) . Conclusion : The expression of OX - 62 and MHC - 鈪，

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