HBV感染者免疫耐受期与免疫清除期PD-1表达变化及意义

发布时间：2018-02-27 16:50

本文关键词： 乙型肝炎病毒慢性乙型肝炎程序性死亡受体-1 程序性死亡配体-1 细胞毒性T淋巴细胞免疫耐受　出处：《河北医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:根据免疫学特点,慢性乙型肝炎病毒(hepatitis B virus,HBV)感染在临床上可以分为三个时期,即免疫耐受期,免疫清除期,免疫控制期或非活动携带状态。目前HBV感染慢性化的确切机制尚不明确。HBV特异性T细胞功能低下可能是HBV感染慢性化的主要原因之一,而负性共刺激信号可导致T细胞功能低下。程序性死亡受体-1(programmed death-1,PD-1)及其配体PD-L1是重要的负性共刺激信号,与CD8~+T细胞失活及病毒持续感染有关。最近的研究表明,在慢性HBV感染者中,病毒特异性CD8~+T细胞表面的PD-1表达水平增加,阻断PD-1/PD-L1信号通路后T细胞功能可得到一定程度恢复。虽然关于HBV感染患者PD-1表达水平和T细胞反应变化的报道很多,慢性HBV感染的自然过程中免疫耐受期与免疫清除期患者PD-1的表达变化规律研究目前少见报道。本研究旨在探讨PD-1在HBV感染者免疫耐受期与免疫清除期的表达变化及意义。方法:选取慢性HBV感染者105例,其中免疫清除期患者55例,免疫耐受期患者50例,所有患者均行肝组织活检,炎症分级为G0~G4,纤维化程度分为S0~S4。健康对照15例。 1所有受试者留取新鲜外周静脉血,密度梯度离心法分离PBMC,用PD-1-FITC抗体进行染色,并以流式细胞仪检测PBMC上PD-1表达水平,同时用HLA-A2抗体进行染色,流式细胞仪行HLA-A2型别鉴定。 2选取HLA-A2阳性的受试者(免疫清除期患者30例,免疫耐受期患者30例,正常人10例),密度梯度离心法分离PBMC,用pentamer-PE、CD3-ECD、CD8-PE-Cy5、PD-1-FITC抗体进行染色,并以流式细胞仪检测HLA-A2阳性的HBV特异性CD8~+T细胞占总的CD8~+T细胞比例及其PD-1表达。结果: 1不同临床分期慢性HBV感染者PBMC及HBV特异性CD8~+T细胞上PD-1的表达变化:免疫耐受组PBMC及HBV特异性CD8~+T细胞上PD-1表达明显高于免疫清除组与正常对照组,差异有统计学意义(P0.05或P0.01)。2不同临床分期慢性HBV感染者HBV特异性CD8~+T细胞表达变化:免疫清除组HBV特异性CD8~+T细胞表达明显高于免疫耐受组与正常对照组,差异有统计学差异(P0.01)。 3 HBV特异性CD8~+T细胞数量与其PD-1表达的相关性:HBV特异性CD8~+T细胞数量与其PD-1表达呈负相关(r=-0.463, P0.01)。 4 HBV特异性CD8~+T细胞上PD-1表达与HBV DNA的关系:按HBV DNA水平将研究对象分为3 log10、3-6 log10和6 log10三组。6 log10组HBV特异性CD8~+T细胞上PD-1表达较3 log10组和3-6 log10组明显增高(F=82.94,P0.01)。相关性分析显示HBV特异性CD8~+T细胞上PD-1表达与HBV DNA呈正相关(r=0.882, P0.01)。 5免疫清除组HBV特异性CD8~+T细胞上PD-1表达与血清ALT水平的相关性:按ALT水平将研究对象分为3倍、3-10倍和10倍正常值三组。结果:三组之间PD-1表达无明显差异(P0.05)。相关性分析显示HBV特异性CD8~+T细胞上PD-1表达与ALT无相关(r=0.085, P0.05)。结论: 1慢性HBV感染者中免疫耐受期患者HBV特异性CD8~+T细胞表达较免疫清除期患者明显减少,提示HBV特异性CD8~+T细胞在清除HBV中发挥重要作用。 2慢性HBV感染者中免疫耐受期患者HBV特异性CD8~+T细胞上PD-1的表达明显高于免疫清除期,提示PD-1的高表达可能影响HBV特异性CD8~+T细胞的活化。 3在慢性HBV感染者中,HBV特异性CD8~+T细胞上PD-1表达与HBV特异性CD8~+T细胞表达呈负相关,说明PD-1通过抑制HBV特异性CD8~+T细胞的活化来影响疾病的进展,PD-1的高表达导致T细胞活化障碍可能是乙型肝炎免疫耐受机制之一。
[Abstract]:Objective: according to the immunological characteristics of chronic hepatitis B virus (hepatitis B, virus, HBV) infection in clinic can be divided into three periods, namely, immunotolerance, immune clearance phase, immune control or inactive carrier state. The exact mechanism of the chronicity of HBV infection is not clear the function of.HBV specific T cells may be low is one of the main reasons for the chronicity of HBV infection, while negative costimulatory signals can lead to T cell dysfunction. Programmed death receptor -1 (programmed death-1 PD-1) and its ligand PD-L1 is an important negative costimulatory signal, CD8 and ~+T cell inactivation and virus infection. Recent studies show that in chronic HBV infection, virus specific CD8~+T cell surface expression level of PD-1 increased to a certain extent, blocking the recovery function of T cells in PD-1/PD-L1 signaling pathway. Although the PD-1 expression level of HBV infection patients And T cell response changes to many reports, the natural process of chronic HBV infection in immune tolerance and immune clearance of PD-1 patients the expression of research is rarely reported. This study aimed to investigate PD-1 in HBV infection of immune tolerance and the immune clearance phase expression and significance.
Methods: 105 cases of chronic HBV infection were selected, including 55 cases of immune clearance phase and 50 cases of immune tolerance stage. All patients underwent liver biopsy, and the grade of inflammation was G0~G4. The fibrosis degree was divided into 15 S0~S4. healthy controls.
1 all subjects received fresh peripheral venous blood. PBMC was isolated by density gradient centrifugation. PD-1-FITC antibody was used to stain. PD-1 expression level on PBMC was detected by flow cytometry. HLA-A2 antibody was used for staining, and HLA-A2 type identification was performed by flow cytometry.
The selection of 2 HLA-A2 positive subjects (30 cases, 30 cases of patients with immune clearance, immune tolerance in patients with 10 cases of normal persons), density gradient centrifugation of PBMC, pentamer-PE, CD3-ECD, CD8-PE-Cy5, PD-1-FITC antibody staining and flow cytometry was used to detect HLA-A2 positive HBV specific CD8~+T cells the proportion of the total CD8~+T cells and the expression of PD-1.
Result:
1 different clinical stages of chronic HBV infection, the expression changes of PBMC and HBV specific CD8~+T cells in PD-1: immune tolerance group PBMC and HBV specific CD8~+T cells on the expression of PD-1 was significantly higher than that in the immune clearance group and normal control group, the difference was statistically significant (P0.05 or P0.01).2 in different clinical stages of chronic HBV infection HBV specific CD8~+T cell expression: the expression was significantly higher than that of immune tolerance group and normal control group group HBV specific CD8~+T cell immune clearance, there was significant difference (P0.01).
The correlation between the number of 3 HBV specific CD8~+T cells and the expression of PD-1: the number of HBV specific CD8~+T cells was negatively correlated with the expression of PD-1 (r=-0.463, P0.01).
The expression of specific CD8~+T cells on PD-1 4 HBV HBV DNA and HBV DNA: according to the level of the research object will be divided into 3 log10,3-6 and 6 log10 log10 three.6 group log10 group HBV specific CD8~+T cells on the expression of PD-1 was 3 log10 and 3-6 log10 groups increased significantly (F=82.94, P0.01). Correlation analysis showed that HBV specific CD8~+T cells on the expression of PD-1 was positively correlated with HBV DNA (r=0.882, P0.01).
5 immune clearance group HBV specific CD8~+T cells on the expression of PD-1 correlated with serum ALT levels: ALT level according to the research object will be divided into 3 times, 3-10 times and 10 times the normal value of the three groups. Results: the three groups had no significant difference between the expression of PD-1 (P0.05). Correlation analysis showed that HBV specific CD8~+T cells in PD-1 no correlation with the expression of ALT (r=0.085, P0.05).
Conclusion:
1 the expression of HBV specific CD8~+T cells in patients with chronic HBV infection in immune tolerance phase is significantly reduced than that in immune clearance phase, suggesting that HBV specific CD8~+T cells play an important role in clearing HBV.
2 the expression of PD-1 on HBV specific CD8~+T cells in patients with chronic HBV infection is significantly higher than that in immune clearance phase, indicating that the high expression of PD-1 may affect the activation of HBV specific CD8~+T cells.
In 3 patients with chronic HBV infection, HBV specific CD8~+T cells on the expression of PD-1 was negatively correlated with HBV specific CD8~+T cells express PD-1 by inhibiting the activation of HBV specific CD8~+T cells to influence the progression of the disease, the high expression of PD-1 leads to activation of T cell disorder may be one of the mechanisms of immune tolerance of hepatitis B.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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