【作者】 刘振芳；

【导师】 孙汉英；

【作者基本信息】 华中科技大学 ， 内科学， 2006， 博士

【摘要】 第一部分再生障碍性贫血小鼠骨髓T细胞表面CD28、CTLA4水平检测及其意义目的:建立再生障碍性贫血小鼠模型。体外观察再障小鼠骨髓T细胞表面CD28、CTLA4的表达变化及其意义。方法建立免疫介导再障小鼠模型,分离再障小鼠和正常组小鼠骨髓单个核细胞(BMMNC),体外利用植物血凝素(PHA,终浓度为15μg/ml)特异性激活T细胞,用双色免疫荧光标记流式细胞术检测正常小鼠和再障小鼠于PHA刺激前后骨髓T细胞表面CD28和CTLA4的表达水平。结果PHA刺激培养48小时后,正常小鼠和再障小鼠骨髓T细胞表面CD28和CTLA4的表达较刺激前均明显升高(P<0.0 1);而且再障小鼠PHA刺激培养前后T细胞CD28的表达均显著高于同期正常小鼠(P<0.0 1),但CTLA4的表达与同期正常小鼠相比虽略有升高,但差异无显著性(P>0.05)。结论再障小鼠骨髓T细胞激活及其激活潜能增强,免疫分子CD28和CTLA4在骨髓中的异常表达可能参与了再障T细胞免疫功能紊乱,或是紊乱的表现之一。第二部分再生障碍性贫血小鼠骨髓淋巴细胞Th1/Th2细胞亚群漂移与转录因子T-bet和GATA-3的调控作用目的:探讨再障小鼠骨髓淋巴细胞的Th1/Th2细胞亚群的漂移与转录因子T-bet和GATA-3的调控作用。方法:分离正常小鼠和再障小鼠的骨髓淋巴细胞,在终浓度为15μg/ml植物血凝素(PHA)条件下培养,用双抗体夹心ELISA方法检测小鼠骨髓淋巴细胞培养上清中Th1类细胞因子IFN-γ和Th2类细胞因子IL-4的水平;用RT-PCR技术检测小鼠骨髓淋巴细胞IFN-γ、IL-4以及转录因子T-bet和GATA-3 mRNA表达强度;Western Blot方法检测T-bet和GATA-3的蛋白表达水平。结果:①再障小鼠骨髓淋巴细胞分泌Th1/Th2类细胞因子INF-γ和IL-4浓度及其在淋巴细胞中mRNA的表达水平均显著高于正常组(P<0.0 1),而IL-4基因和蛋白水平明显低于正常组(P<0.0 1);②再障小鼠骨髓淋巴细胞转录因子T-bet基因和蛋白水平显著高于正常组(P<0.0 1),而GATA-3基因和蛋白水平明显低于正常组(P<0.0 1);相关性分析显示,再障小鼠IFN-γ浓度与T-bet蛋白水平呈正相关,与GATA-3无相关性,而L-4浓度与GATA-3呈正相关,与T-bet呈负相关。结论:再障小鼠骨髓出现T细胞向Th1亚群漂移现象,导致机体Th1细胞的优势应答状态,从而诱导细胞毒性T细胞对造血干细胞的细胞毒作用,而T-bet表达增强与GATA-3低表达可能是再障小鼠骨髓Th1优势分化的重要原因。第三部分融合蛋白CTLA4Ig对再生障碍性贫血小鼠骨髓T细胞功能的作用研究目的:观察CTLA4Ig对再生障碍性贫血小鼠骨髓T细胞增殖、Th1和Th2类细胞因子表达水平以及T细胞对靶细胞的细胞毒活性的影响。方法:利用同种异体来源的树突状细胞(DC)作为抗原和抗原提呈细胞,在终浓度为25μg/ml的丝裂霉素C处理后作为刺激细胞,尼龙毛方法分离正常小鼠和再障小鼠骨髓的T细胞作为反应细胞,进行单向混合淋巴细胞培养(MLR),同时加入终浓度为10μg/ml的CTLA4Ig共培养,MTT比色法检测CTLA4Ig对T细胞增殖的影响,ELISA方法检测MLR培养上清Th1和Th2类细胞因子水平变化;体外诱导CTL,乳酸脱氢酶试验检测CTLA4Ig对再障骨髓CTL细胞毒活性的影响。结果:CTLA4Ig可以在体外明显抑制再障小鼠过度的T细胞增殖,并使其降至正常水平;可以下调Th1类细胞因子,上调Th2类细胞因子,诱导再障小鼠骨髓Th1/Th2失衡向Th2的转换,使再障Th1异常优势应答状态恢复正常;抑制再障小鼠CTL细胞的杀伤活性。结论:CTLA4Ig能够在体外抑制再障小鼠骨髓T细胞的增殖、诱导Th1/Th2的转换,恢复再障Th1异常优势应答状态,抑制骨髓CTL的杀伤活性,从而恢复骨髓造血。更多还原

【Abstract】 Part ⅠExpressions of CD28 and CTLA4 on T cells from bone morrow in aplastic anemia mice.Objective: To investigate the expressions and significance of CD28 and CTLA4 on T cells in bone marrow of aplastic anemia (AA) mice,Methods: In vitro bone marrow mononuclear cells (BMMNCs) were activated through being incubated with PHA (15μg/ml). The CD28 and CTLA4 expressions on T cells incubated with or without PHA were analyzed by two color flow cytometry. The expressions of CD28 and CTLA4 significantly increased after PHA stimulation. In the AA mice, the expressions of CD28 with or without PHA stimulation were both higher than that in the normal mice (P<0.01 and P<0.01), but the expressions of CTLA4 with or without PHA stimulation had both no significant difference compared with that in the normal mice (P>0.05 and P>0.05). In the AA mice, there were more activation and activated potential of T cell than the normal, and the abnormal expressions of CD28 and CTLA4 maybe participate in immunological disorder mediated by T cell. Part ⅡT cell specific transcription factors T-bet and GATA-3 contribute to shifted development of T help cell toward type I from bone marrow in aplastic anemia (AA) miceObjective: To investigate the T helper cell predominant differentiation in aplastic anemia (AA) mice and to explore the modulation of T cell specific transcription factors T-bet and GATA-3.Methods: Established model of AA mice. Lymphocytes were isolated from bone marrow incubated with phytohemagglutinin (PHA)(15μg/ml) in vitro. Cytokines IL-2, IFN-γ, IL-4 and IL-10 concentration in bone marrow were detected by ELISA. The gene expression levels of transcription factor T-bet/GATA-3 were assayed by RT-PCR.Results: In AA mice, the levels of IL-2 and IFN-γ were markedly increased but IL-4 and IL-10 were markedly decreased than normal mice. And the gene and protein expressions of T-bet in AA mice were much higher than normal, but GATA-3 were lower than normal.Conclusion: In AA mice, there is a shifted development of Th toward to Th1, which maybe involved in induction of T-cells cytotoxic to hematopoietic stem cell. Furthermore, type I shift of Th cause by the level of T-bet was significantly over expressed and GATA-3 decreased. Part ⅢEffect of CTLA4Ig on T cells which from bone marrow in aplastic anemia (AA) mice in vitroObjective: To study the effect of CTLA4Ig on T cells which from bone marrow in aplastic anemia mice in vitro.Methods: T cells from bone marrow of normal and AA mice (as reaction cells) with same amount of mitocin-C treated DC (as stimulation cells) were co-cultured for 5 days in the presence CTLA4Ig. MTT colorimetry was used to detect allogeneic T cells proliferation. Using ELISA assay to detect the level of allogeneic response T cells cytokine: IFN- γ and IL-4. Co-cultured the above allogeneic response T cells (as stimulation cells) with DC (as reaction cells), using LDH Cytotoxicity Assay Kit detect the cytotoxic activity of CTL.Result: In aplastic anemia the proliferation response of T cells induced by DC and in presence CTLA4Ig was significantly lower than which without CTLA4Ig, the level of INF- γ was markedly decreased and the level of IL-4 markedly increased by CTLA4Ig, the cytotoxic activity was markedly decreased by CTLA4Ig in vitro.Conclusion: In aplastic anemia, CTLA4Ig could inhibit T cells proliferation, down-regulate the Th1 cytokines and up-regulate the Th2 cytokines, and inhibit the cytotoxic activity in vitro, so that induced T cell anergy by blocking B7/CD28pathway.更多还原