【作者】 吴静；

【导师】 张学光； 顾国浩；

【作者基本信息】 苏州大学 ， 医学免疫学， 2014， 硕士

【摘要】 【背景】围绝经期是指从接近绝经出现与绝经有关的内分泌、生物学和临床特征起至最后一次月经后一年内的时期。处于围绝经期的妇女由于卵巢功能衰退，神经、内分泌及免疫系统均会出现紊乱而出现一系列相应的临床症状。机体的免疫系统通过正性和负性共刺激信号的动态平衡，保持机体免疫状态的稳定。正性共刺激信号(如B7/CD28)决定了T淋巴细胞是否能识别相应抗原来达到免疫的完全激活，相反，T淋巴细胞表达的负性共刺激分子(B7/CTLA-4)则可以调节免疫反应。PD-1（Programmed death-1）是一个新近发现的共刺激分子，PD-1通过与其两个配体PD-L1和PD-L2作用而抑制T、B细胞的活化及细胞因子的产生，在维持机体免疫耐受上发挥至关重要的作用。同时，PD-1分子也与一系列疾病的免疫病理发生发展密切相关。研究发现雌激素（E2）可能通过上调T细胞PD-1的表达发挥免疫抑制功能；然而阻断PD-1/PD-L1通路后雌激素治疗可以增加T细胞免疫活性，提示雌激素可能是PD-1分子表达的调节因素之一。临床上大多集中研究围绝经期妇女的激素水平，对该期妇女的免疫功能方面的研究相对较少。对围绝经期妇女共刺激分子表达与雌激素水平的相关性也未见报道。因此，深入研究围绝经期妇女共刺激分子表达及其与雌激素水平的关系对于了解围绝经期妇女免疫学功能下调的机制具有重要作用。也可为围绝经期诊断和预后判断提供有临床应用意义的生物学标记。【目的】研究围绝经期和绝经后期妇女外周血单个核细胞表面和T细胞表面CD28、CTLA-4、PD-1和PD-L1的表达，旨在了解围绝经期和绝经期妇女相关免疫细胞表面生物学标志表达的变化情况，结合血清雌激素变化情况，探讨围绝经期和绝经后期妇女内分泌、免疫系统之间的关系及临床意义。【方法】采用Taqman探针实时荧光定量PCR法、流式细胞术及化学发光法检测70例围绝经期、40例绝经后期和30例育龄期组外周血单个核细胞表面共刺激分子CD28、CTLA-4、PD-1、PD-L1mRNA的表达，T细胞表面共刺激分子及T淋巴细胞亚群表达，并与其血清雌二醇（E2）、孕激素（Pg）、卵泡刺激素(FSH)和黄体生成素(LH)水平作综合分析。【结果】（1）、与育龄期组比较，E2、Pg在围绝经期组和绝经后期组显著下降（P<0.05）；FSH/LH比值在绝经后期组和围绝经期组显著高于育龄期组（P<0.05）；（2）、CD28mRNA组间表达绝经后期组最高（6.84±0.56），育龄期组最低（6.37±0.54），差异有统计学意义（F=5.729，P<0.05）；CTLA-4mRNA间表达量绝经后期组最高（6.80±1.01），围绝经期组最低（5.32±0.90），差异有统计学意义（F=28.594，P<0.01）；PD-1mRNA组间表达量绝经后期组最高（5.46±0.68），育龄期组最低（4.43±0.89），差异有统计学意义（F=12.222，P<0.01）；PD-L1mRNA组间表达量绝经后期最高（5.07±0.85），育龄期组最低（3.82±0.68），差异有统计学意义（F=24.825，P<0.01）。（3）、CD3+、CD4+相对表达率及绝对值、CD4+/CD8+比值在三组间比较均有统计学差异（P<0.05）。经两两比较发现：与育龄期组比较，围绝经期和绝经后期组的CD3+、CD4+相对表达率及绝对值以及CD4+/CD8+比值均有不同程度地显著下降。T细胞表面CD28表达量在三组间比较均有统计学差异，其中围绝经期和绝经后期组均显著低于育龄期组；CD28+/CTLA-4+比值在绝经后期组显著低于围绝经期组和育龄期组；PD-1的表达在三组间比较有显著的统计学差异，围绝经期及绝经后期组均显著低于育龄期组。（4）、相关性分析发现：PD-1、PD-L1mRNA的表达与E2呈不同程度负相关（r值-0.207、-0.353，P值0.031、<0.001）；CD3+、CD4+T细胞表达量与E2呈显著的正相关（r=0.256、0.246，P=0.007、0.005）；与年龄呈显著的负相关（r=-0.387、-0.456，P<0.001）；CD4+PD-1+与E2呈显著的正相关（r=0.550，P<0.001）；CD8+PD-1+与E2呈显著的正相关（r=0.554，P<0.001）。【结论】1、围绝经期和绝经后期妇女存在T淋巴细胞亚群的紊乱，CD3+、CD4+和CD8+T细胞相对表达率及绝对值均下降，提示其T细胞免疫功能失调。2、外周血单个核细胞表面共刺激分子CD28、PD-1、PD-L1mRNA表达显著升高，CTLA-4显著降低，且T细胞表面CD28、CTLA-4、和PD-1的表达量均出现了不同程度的降低，提示共刺激分子表达异常可能与免疫功能失调有关。3、CD3+T、CD4+T细胞表达量、CD4+PD-1+、CD8+PD-1+与E2呈显著的正相关，提示雌激素可能调控PD-1分子表达,下调围绝经期和绝经期妇女免疫功能。综上所述，围绝经期及绝经后期女性内分泌激素异常、外周血单个核细胞表面共刺激分子mRNA表达异常及T细胞表面共刺激分子和T淋巴细胞亚群的表达量异常与其免疫力下降密切相关。通过以上这些分析可更加完善地判断其生理状况，进一步了解该期女性的免疫功能状态。更多还原

【Abstract】 Objective To analyze the CD28,CTLA-4,PD-1and PD-L1expression on peripheralblood mononuclear cells and T cells of perimenopausal and postmenopausal women. Inattempt to investigate the relevance and significance of the immune function and changesin serum estrogen.Methods The mRNA expression of CD28,CTLA-4, PD-1and PD-L1on peripheralblood mononuclear cells were detected by Taqman probe real-time quantitative PCR, andthe levels of CD28,CTLA-4and PD-1on T cells and T lymphocyte subsets weredetermined by Flow Cytometry, including70cases perimenopausal,40casespostmenopausal women and30cases normal control(NC). And estradiol (E2),progesterone(Pg),follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels in serum forcomprehensive analysis.Results There were statistically significant difference with the expression level ofCD28mRNA(F=5.729, P <0.05) in different group,with the highest expression level inthe postmenopausal groups (6.84±0.56) and with the lowest in healthy control group(6.37±0.54). There were statistically significant difference with the expression level ofCTLA-4mRNA (F=28.594, P <0.01) in different groups, with the highest expression levelin the postmenopausal group(6.80±1.01)and with the lowest in perimenopausal group(5.32±0.90). There were statistically significant difference with the expression level ofPD-1mRNA (F=12.222, P <0.01) in different group,with the highest expression level inthe postmenopausal group (5.46±0.68) and with the lowest in healthy control group(4.43±0.89). There were statistically significant difference with the expression level of PD-L1mRNA (F=24.825, P <0.01) in different groups,with the highest expression level in thepostmenopausal groups (5.07±0.85) and with the lowest in healthy control group (3.82±0.68).There were significant positive correlation of the expression levels of PD-1mRNA and the level of E2(r=-0.207,P=0.031), and there were significant negative correlation ofthe expression level of PD-L1mRNAand the level of E2（r=-0.353,P<0.001）.There werestatistically significant difference with the expression levels of CD3+and CD4+Tcells andthe ratio of CD4+/CD8+in the three groups. All of the levels in perimenopausal andpostmenopausal groups were lower than NC. There were statistically significant differencewith the expression levels of CD28on T cells, and in the perimenopausal andpostmenopausal groups were significantly lower than NC. The ratio of CD28+/CTLA-4+inthe postmenopausal group was much lower than perimenopausal group and NC. Therewere statistically significant difference with the expression levels of PD-1on T cells, andin the perimenopausal and postmenopausal groups were significantly lower than NC. Therewere significant positive correlation of the expression levels of CD3+and CD4+Tcells andthe level of E2(r=0.256,0.246.P=0.007,0.005).There were significant positive correlationof the expression levels of CD3+and CD4+T cells and the age(r=-0.387,-0.456.P<0.001).PD-1on CD4and CD8T cells show significant positive correlation with the level ofE2(r=0.550,0.554.P<0.001).Conclusion1. The relative expression and absolute value of CD3+,CD4+and CD8+T cells wereall decreased in the perimenopausal and postmenopausal women. It was implied that thewomen exist T cell immune dysfunction.2. The mRNA expression of CD28,CTLA-4, PD-1and PD-L1on peripheral bloodmononuclear cells were shown disorderd. The transcriptional change provides basicinformation for further study the interaction of co-stimulatory molecules, estrogen andimmune function.3. There was a significant positive correlation between the amount of CD3+T, CD4+T,PD-1and the level of E2. It implied that estrogen may be regulating the expression ofPD-1molecule and reduced perimenopausal and postmenopausal women immunefunction.更多还原