慢性乙肝病毒感染不同免疫状态患者树突状细胞体外刺激后介导的 T淋巴细胞功能影响的研究

Functions of T lymphocytes Mediated by in vitro Dendritic Cells of Chronic HBV Infection Patients in Different Immune States:a Clinical Research

目的探讨通过补肾解毒和健脾解毒两种不同药物血浆体外作用慢性乙型肝炎(chronic hepatitis B,CHB)患者树突状细胞(dendritic cells,DCs)后对DCs功能的改善,比较哪种药物血浆更能促进DCs激活T淋巴细胞的功能提高。方法选择门诊CHB患者30例,根据其免疫状态,分为免疫耐受期组15例,免疫清除期组15例,抽取每例患者外周血60mL,分离培养出成熟的DCs,在培养第7天加入含补肾解毒(六味甘露饮)与健脾解毒(四君甘露饮)的复方中药血浆促进DCs成熟,另设无药物血浆作为对照。培养至第9天,将DCs负载HBVcore18-27核心肽后与自身的T淋巴细胞共培养72h,收集T淋巴细胞,应用流式细胞技术检测T细胞表面分子CD3、CD28、CD4、CD8及程序型坏死因子-1(programmed death-1,PD-1)的表达水平。结果与无中药血浆的共培养比较,免疫耐受期组和免疫清除期组的患者经补肾解毒和健脾解毒药物血浆干预后的DCs与T细胞共培养,在一定程度上都能提高CD3、CD4、CD28的表达水平,降低CD8、PD-1的表达水平,差异均有统计学意义(P<0.05),且补肾解毒药物血浆对提高免疫耐受组期患者CD28分子表达率、减低T细胞表面PD-1分子的表达率,效果均优于健脾解毒血浆的干预,差异均有统计学意义(P<0.05)。结论通过补肾解毒、健脾解毒药物血浆对DCs体外干预,并与自身T淋巴细胞共培养的方法,能够促进CHB患者DCs激活T细胞的功能提高和T细胞表面分子的表达,且补肾解毒法对慢性乙型肝炎病毒(hepatitis B virus,HBV)感染免疫耐受期患者影响更为显著。

Objective To explore the improvement of dendritic cells＇ （DCs） functions in chronic hepatitis B （CHB） patients by two different drugs plasma, i.e., Shen supplementing and detoxification （SSD） and Pi invigorating and detoxification （PID）, thus comparing which method was more effective to activate DCs to improve T lymphocyte functions. Methods Totally 30 CHB outpatients were recruited. They were assigned to the immune tolerant group and the immune clearance group, 15 in each group. To- tally 60 mL peripheral blood was extracted to isolate and develop mature DCs. Chinese compound containing （Liuwei Ganlu Syrup for SSD） and （Sijun Ganlu Syrup for PID） plasma were added to promote the maturation of DCs on the 7th day. Besides, non-drug plasma was taken as the control. On the ninth day, HBVcore18 -27 loaded core peptide and its own T lymphocyte were co-cultivated for 72 h. Then T lympho- cytes were collected. The expression levels of CD3, CD28, CD4, and CD8, programmed death-1 （PD-1） were detected using flow cytometry. Results Compared with non-drug plasma, the expression levels of CD3, CD4, and CD28 could be improved, and the expression levels of CD8 and PD-1 could be reduced by the two methods, showing statistical difference （P 〈0.05）. Besides, SSD containing plasma showed better effect in improving the molecular CD28 expression rate, and reducing the molecular PD-1 expres- sion rate on the T cell surface, showing statistical difference when compared with that of PID containing plasma （P 〈0.05）. Conclusions In vitro intervention of DCs by SSD and PID containing plasmas com- bined co-cultivation of its own T lymphocytes could promote the activation of DCs to improve the function of T cells and the expression of T cell surface molecules. Besides, SSD showed more significant effect on infection immune of HBV patients in the tolerance stage.