【作者】 张旻；

【导师】 龚国忠；

【作者基本信息】 中南大学 ， 内科学， 2012， 博士

【摘要】 第一部分慢性乙型肝炎患者外周血淋巴细胞表面的PD-1表达及其影响因素的研究目的研究慢性乙型肝炎患者外周血外周血单个核细胞、CD4+T、CD8+T淋巴细胞表达PD-1的情况以及影响PD-1表达的可能的因素。方法密度梯度离心法分离63例慢性乙型肝炎患者(其中包括HBeAg(+)患者32例,HBeAg(一)患者31例)、35例乙肝病毒健康携带者、18例健康人外周血单个核细胞、CD4+T、CD8+T淋巴细胞中表达PD-1的百分比,分析慢性乙型肝炎患者、乙肝病毒健康携带者及健康人PD-1表达的差异；观察PD-1表达与HBV感染者ALT水平、乙肝e抗原、病毒复制指标HBV-DNA水平之间的关系。结果HBV健康携带组外周血单个核细胞、CD4+T淋巴细胞、CD8+T淋巴细胞表面PD-1表达率最高；CHB组次之；健康对照组最低。HBV健康携带组外周血单个核细胞、CD4+T淋巴细胞上PD-1表达显著高于健康对照组及CHB组,CHB组显著高于健康对照组(P<0.05)。而CD8+T淋巴细胞上PD-1表达只在健康对照组和HBV健康携带组之间比较差异显著(P<0.05),其他组间比较均无统计学差异。CHB患者外周血单个核细胞、CD4+T、CD8+T淋巴细胞上表达PD-1百分比与血清ALT及HBV-DNA水平无相关性。CHB患者eAg是否阳性对PD-1在外周血单个核细胞、CD4+T细胞、CD8+T细胞表面表达率有一定影响：HBeAg(+)组和HBeAg(-)组外周血单个核细胞表面表达PD-1比率分别为：25.88±20.09%和15.39±10.64%,较健康对照组(10.85±4.45%)明显升高,HBeAg(+)组和健康对照组比较,差异有统计学意义(P<0.05)；HBeAg(+)组PD-1表达比率显著高于HBeAg(-)组,P<0.01。HBeAg (+)组、HBeAg(-)组、健康对照组CD4+T淋巴细胞表面表达PD-1表达率分别为：9.15±6.46%、8.65±6.84%、2.37±1.16%,HBeAg(+)组及HBeAg(-)组健与康对照组CD4+T淋巴细胞表面PD-1表达率比较,差异均有统计学意义,P<0.01。HBeAg(+)组、HBeAg(-)组、健康对照组CD8+T淋巴细胞表面PD-1表达率分别为：3.71±2.79%、3.23±2.39%、1.40±0.31%,HBeAg(+)组及HBeAg(-)组健与康对照组CD8+T淋巴细胞表面PD-1表达率比较,差异有统计学意义,P<0.05。结论CHB患者外周血单个核细胞、CD4+T淋巴细胞上PD-1表达显著高于健康人,又明显低于HBV健康携带者。PD-1表达可能与HBV感染者的免疫状态相关。血清HBeAg可以明显影响CHB患者外周血T单个核细胞表面PD-1的表达,是影响其PD-1表达的重要因素。第二部分淋巴细胞系Molt-4细胞PD-1启动子区域甲基化水平和PD-1表达的关系的研究目的以T淋巴细胞株Mo1t-4细胞为模型,探讨甲基化抑制剂5-杂氮胞苷(5-Zac)对淋巴细胞表面程序性死亡受体-1(PD-1)基因启动子的去甲基化作用及其诱导的PD-1基因表达的改变,并进一步研究去甲基化作用与PD-1基因表达之间关系。方法以不同浓度的5-Zac(0μmol·L-1、5μmol·L-1、10μmo1·L-1)作用于体外培养的molt-4细胞72h,流式细胞术(FCM)检测细胞表面表达PD-1的Molt-4细胞比例和细胞凋亡率；反转录-聚合酶链反应(RT-PCR)检测5-Zac作用后PD-1基因mRNA的转录水平；亚硫酸氢钠处理各组Molt-4细胞DNA, PCR扩增PD-1启动子基因片段,转化感受态大肠杆菌,挑克隆测序,检测扩增的PD-1启动子片段甲基化状态。结果0μmol·L-I、5μmol·L-1、10μmol·L-1的5-氮杂胞苷作用于molt-4细胞72h后,PD-1在细胞表面的表达率分别为：1.13±0.01%；18.96±1.87%；63.09±6.25%,并呈现浓度依赖性；PD-1基因mRNA表达量显著增加；细胞凋亡检测结果显示与未处理组相比,加5μmol·L-1、10μmo1·L-1的5-氮杂胞苷处理72h后molt-4细胞的凋亡率显著增加,三组凋亡率分别为：1.9%+0.06%；8.98%+1.36%；24.5%+3.68%,有显著性差异(P<0.01)；三组DNA亚硫酸氢钠测序结果表明,加入甲基化抑制剂5-Zac处理后,PD-1启动子上-601bp和d-553bp CG点去甲基化程度明显增高。结论甲基化抑制剂5-Zac可导致体外培养的T淋巴细胞系Molt-4细胞表面程序性死亡受体-1(PD-1)表达显著增加,PD-1基因mRNA表达增加,细胞凋亡率增高,这种增高可能与PD-1基因启动子区域出现的低甲基化有关。第三部分慢性乙型肝炎患者外周血单个核细胞PD-1基因启动子区域甲基化状态及影响因素目的探讨慢性乙型肝炎患者外周血单个核细胞上程序性死亡受体-1表达和PD-1启动子区域甲基化水平的关系,以及HBeAg对慢性乙型肝炎患者外周血单个核细胞表面PD-1基因启动子区域甲基化状态的影响方法筛选健康对照组10例、慢性乙型肝炎患者HBeAg阳性者10例、慢性乙型肝炎患者HBeAg阴性者10例,实时定量聚合酶链反应(real-time RT-PCR)检测外周血单个核细胞中PD-1基因的mRNA表达水平；亚硫酸氢钠处理PBMCs细胞DNA, PCR扩增PD-1启动子基因片段,转化感受态大肠杆菌,挑克隆测序,检测扩增的PD-1启动子片段甲基化状态。结果与正常对照组(0.10±0.023)比较,慢性乙型肝炎患者外周血单个核细胞中PD-1mRNA表达水平明显升高。在慢性肝炎患者中,外周血单个核细胞上PD-1表达在HBeAg阳性患者(0.44±0.046)中显著高于HBeAg阴性患者(0.18±0.014)。PD-1启动子上多个CpG点(-601、-553、-538、-483、-463、-317)甲基化程度与PD-1在单个核细胞上的表达呈负相关；PD-1启动子上-553bp、-538、-483CpG位点甲基化程度与血清中e抗原可能存在相关性。结论PD-1基因启动子区域甲基化水平可能影响慢性乙肝患者外周血单个核细胞上PD-1表达；其基因启动子甲基化状态可能受到乙肝病毒e抗原影响。更多还原

【Abstract】 Part one:The expression and influence factors of programmed death receptor1on peripheral blood mononuclear cells of chronic hepatitis B virus-infected patientsObjective To observe the expression variance of Programmed death one (PD-1) on PBMCs and CD4+T/CD8+T lymphocytes in Chronic Hepatitis B patients and further investigate The relationship between the probable influence factors and the PD-1expression on peripheral blood mononuclear cells (PBMC) in CHB Patients.Methods A total of116subjects, including63patients with chronic hepatitis B(CHB),35healthy hepatitis b virus carriers,18healthy blood donators were enrolled. The expression of PD-1, CD4and CD8on the peripheral blood mono-nuclear cells (PBMCs) were detected by FCM; The serum HBV markers, HBV DNA load and liver function were also measured. The difference of PD-1expression on PBMCs, CD4+Tand CD8+T lymphocytes were analyzed in chronic hepatitis B and hepatitis B virus healthy carriers and healthy people enrolled in this study. The relationship between the HBV-DNA, ALT level and hepatitis B e antigen were further observed in CHB patients.Results Taken the PD-1expression in normal controls as a baseline level, the expression of PD-1on PBMCs and CD4+TT lymphocytes in CHB patients and hepatitis B virus healthy carriers was significantly increased; hepatitis B virus healthy carriers was much higher than CHB patients. PD-1expression on CD8+T lymphocytes had significantly difference only between hepatitis B virus healthy carriers and normal controls, but no significant difference between other groups. In CHB patients, the PD-1expression in PBMCs from patients with HBeAg positive in serum was much higher than that from those with HBeAg negative in serum, And the PD-1expression level not correlated with serum HBV DNA load and serum ALT level.Conclusion The PD-1expression level on CHB patients is much higher than normal controls but significantly lower than hepatitis B virus healthy carriers. PD-1expression on peripheral blood mononuclear cells (PBMC) may Associated with the immune state in chronic HBV infectors. Long-term exposure to HBV e antigens in CHB patients may impact the PD-1expression level on PBMCs. Part two:Effects of5-Zac on demethylation pattern of the PD-1gene in promoter region and PD-1expression in a human T lymphocyte cell lineObjective To observe the DNA demethylation of the PD-1promoter caused by5-azacytidine (5-azac) in Molt-4cells (T lymphocyte cell line) and further investigate the relationship between the expression of PD-1and DNA demethylation.Methods The Molt-4cells were cultured in medium containing different concentrations of5-azac(0,5,10Umol/L)for72h;The expression of PD-1in Molt-4cells and the apoptosis were detected by FCM; the mRNA transcription level of PD-1was detected by RT-PCR;Molt-4cell DNA in all groups were treated by sodium bisulfite; The PD-1promoter fragment was amplified by PCR,these amplification fragments were transformed into E. coli. Positive clones were selected for equencing, methylation status of the fragments of PD-1promoter was examined.Results5-azac(1.13%±0.01%) treated cells was found more lower than that in both5μmol/L and10μmol/L5-azac treated cells (18.96%±1.87%,63.09%±6.25%,P<0.05), and they showed concentration-dependent (P<0.01). Cells apoptosis rate and PD-1mRNA expression were also observed increased significantly with5-azac treating. The results of bisulfite genomic sequencing showed that Demethylation probability of CG points on-601bp and-553bp were significantly increased in the5-Zac treated cells compared with the untreated cells.Conclusion5-azac inhibits cell grouth in human lymphoid cell series Molt-4by inducing PD-1gene expression and promoter demethylation. PD-1gene promoter demethylation may be one of the important mechanisms for PD-1increased expression in5-azac treated Molt-4cells. Part three:The methylation pattern of PD-1promoter region and its influence factors in peripheral blood mononuclear cells of chronic hepatitis B virus-infected patientsObjective To observe the relationship between the expression of the Programmed death one (PD-1) and DNA demethylation pattern of the PD-1promoter on PBMCs in Chronic Hepatitis B patients and further investigate the effect of hepatitis B virus e antigen on methylation pattern of PD-1promoter region in peripheral blood mononuclear cells (PBMC) of CHB Patients.Methods20patients with chronic hepatitis B(CHB), including10HBeAg positive and10HBeAg negative,18healthy blood donators as control were enrolled. The present study detected the correlation between the PD-1mRNA level in the peripheral blood mononuclear cells of the chronic hepatitis B patients and serum HBeAg levels, then determined the methylation of the promoter region of the PD-1gene using sodium bisulfite sequencing, so as to investigate the effect of HBeAg on PD-1expression and methylation of promoter region in peripheral blood mononuclear cells of chronic hepatitis B patients, and the relationship between them.Results the PD-1mRNA level of the peripheral blood mononuclear cells in the normal control group was (0.10±0.023), which was significantly lower than those in the HBeAg (+) group (0.44±0.046)and the HBeAg (-) group (0.18±0.014)(P<0.05), and the expression rate was higher in the HBeAg (+) group than that in the HBeAg (-) group (P0.05). the correlation analyses between the PD-1expression and the probability of methylation in each locus showed that the probabilities of methylation at-601,-553,-538,-483,-463and-317were correlated with the PD-1expression (P<0.05), Further comparison of the difference of the methylation status of the methylated loci in the promoter of the PD-1gene in the normal control, HBeAg (-) and HBeAg (+) groups demonstrated that the methylation levels at the CpG loci of-553bp,-538and-483were gradually declined in the normal control, HBeAg (-) and HBeAg (+) groups, and significant difference was observed among groups (P<0.05).Conclusion the methylation state of some CpG loci in the promoter region of the PD-1gene is associated with the PD-1expression, and the effect of HBeAg on the PD-1expression in the peripheral blood mononuclear cells of chronic hepatitis B patients may be related to the impact on the demethylation of the CpG loci in some binding sites of the promoter and transcription factor of PD-1.更多还原