补肾解毒法与健脾解毒法对HBV慢性感染不同阶段患者外周血树突状细胞介导的T淋巴细胞功能影响的研究
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摘要
目的：
拟运用HBV慢性感染过程不同阶段的患者外周血DCs与T淋巴细胞体外共培养的细胞模型和中药血浆药理实验方法,比较补肾解毒、健脾解毒哪种方法更能促进DCs活化特异性T淋巴细胞,哪种方法是提高HBV慢性感染后免疫细胞功能、免疫学应答恢复,治疗慢性乙型肝炎的主要方法,为临床提高中医治疗慢乙肝的疗效提供客观依据、治疗思路和研究基础。
方法：
本研究通过前后抽取HBV慢性感染免疫耐受期和免疫清除期共16例患者外周血80ml,另设健康对照组8例,分离培养出成熟的树突状细胞,在培养过程中采用补肾解毒与健脾解毒复方中药药物血浆分别对DCs进行体外干预,并负载HBV核心蛋白,然后与自身的T淋巴细胞共培养72小时,应用流式细胞技术检测T细胞表面分子CD3、CD28、 CD4、CD8及PD-1的表达水平,并用ELISA试剂盒检测T细胞上清液中IFN-γ分泌水平。比较哪种方法干预对免疫细胞功能的恢复影响更大。
结果：
1.干预前的T淋巴细胞(未经中药血浆干预也未与DCs共培养的T细胞)上CD4、CD28的表达水平,健康人组明显高于免疫耐受组和免疫清除组,而以免疫耐受组的患者表达最低,差异有统计学意义(p<0.05)。CD8、PD-1的表达健康人组明显低于免疫耐受组和免疫清除组,其中又以免疫耐受组的表达最高,差异有统计学意义(p<0.05)。CD3的检测结果健康人和免疫清除期组患者差异无显著意义(p>0.05),但耐受期组患者表达明显低下(p<0.05)。上清液中分泌的IFN-γ水平在健康人和免疫清除期组患者差异无显著意义(p>0.05),在耐受期组患者分泌水平明显低下(p<0.05)。
2.在免疫耐受组和免疫清除组的HBV慢性感染患者经补肾解毒(六味甘露中剂量)和健脾解毒(四君甘露小剂量)药物血浆干预后的DC与T细胞共培养,检测其T细胞表面分子的表达率。两种方法在一定程度上都能提高CD3、CD4、CD28的表达水平和上清液中分泌的IFN-γ水平,降低CD8、PD-1的表达水平。
结论：
1.慢性HBV感染免疫清除期和免疫耐受期患者存在T淋巴细胞功能的缺陷,以免疫耐受期患者细胞免疫功能是最低的。
2.通过补肾解毒、健脾解毒药物血浆在一定程度上能够促进HBV慢性感染患者树突状细胞的成熟,使得DCs激活T细胞的功能明显提高,促进T细胞表面分子的表达,并且补肾解毒法对慢性HBV感染免疫耐受期患者细胞免疫学功能的影响更为显著。
Objective:Using HBV chronic infection process different stages of the patients peripheral blood DCs and T lymphocytes of in vitro cultured cells model and traditional Chinese medicine pharmacology experiment method of plasma,comparisoning bushenjiedu and jianpijiedu which method can promote detoxification of DCs activation specific T lymphocytes, Which method is to improve the HBV infection after chronic immune cells function, immunological responses recovery, and the treatment of chronic hepatitis b main method, which for clinical improving the curative effect of TCM therapy for chronic hepatitis b provides an objective basis, treatment idea and research foundation.
Method:This study by extracting HBV chronic infection immune tolerance period and immune clearance phase16patients peripheral blood80ml. Another set8cases of healthy people as control group, To cultivate a separation of mature dendritic cells, in the process of cultivation using filling kidney detoxify and spleen detoxification compound Chinese medicine drugs respectively in plasma DCs intervention, and load HBV core protein, then with own T lymphocytes trained72hours, Application flow cytometry technical inspection T cells molecular CD3, surface, CD8, CD28CD4and PD-1expression, And ELISA test kit on T cells in the fluid of qing IFN-y secretion levels. comparisoning which kind of method is intervention on the immune cells function restore much more influence.
Results:
1.Before the intervention of the CD4+T lymphocytes, CD28+expression level, healthy human group was significantly higher than the immune tolerance group and immune clearance group, and group of patients with immune tolerance to express the minimum(p<0.05). CD8+, PD-1expression of healthy people obviously lower than immune tolerance group and immune clearance group, which the highest expression of immune tolerance(p<0.05), CD3+results of the health and immune clearance phase groups of patients, no significant difference (p>0.05), but the resistance stage patients with low obvious group expression(p<0.05). Clear liquid secretion on IFN-γ level in health and immune clearance phase groups of patients, no significant difference (p>0.05), in tolerance period groups of patients secretion level obviously low (p<0.05).
2.The immune tolerance group and immune clearance group of HBV chronic infection by the spleen and kidney patients with detoxification detoxification medicine after intervention of DC and plasma T cells of trained, testing the T cells surface of molecular expression rate. The two methods in a degree, can improve CD3+, CD4+, CD28+expression level and clear liquid on the secreted IFN-γ level, reduce the CD8+, PD-1expression level.
Conclusion:
1. Chronic HBV immune clearance phase and immune tolerance were present period T lymphocyte function of the defect, immune-tolerant phase in patients with cellular immune function is the lowest.
2. Through the filling kidney detoxification, invigorating spleendetoxification medicine in a certain extent plasma can promote chronic HBV infection were the mature dendritic cells, making the DCs activate t-cells function improved obviously, promote T cells the expression of molecular surface, and filling kidney detoxification method to chronic HBV immune tolerance of the influence of the patient with cells immunological function is more significant.
拟运用HBV慢性感染过程不同阶段的患者外周血DCs与T淋巴细胞体外共培养的细胞模型和中药血浆药理实验方法,比较补肾解毒、健脾解毒哪种方法更能促进DCs活化特异性T淋巴细胞,哪种方法是提高HBV慢性感染后免疫细胞功能、免疫学应答恢复,治疗慢性乙型肝炎的主要方法,为临床提高中医治疗慢乙肝的疗效提供客观依据、治疗思路和研究基础。
方法：
本研究通过前后抽取HBV慢性感染免疫耐受期和免疫清除期共16例患者外周血80ml,另设健康对照组8例,分离培养出成熟的树突状细胞,在培养过程中采用补肾解毒与健脾解毒复方中药药物血浆分别对DCs进行体外干预,并负载HBV核心蛋白,然后与自身的T淋巴细胞共培养72小时,应用流式细胞技术检测T细胞表面分子CD3、CD28、 CD4、CD8及PD-1的表达水平,并用ELISA试剂盒检测T细胞上清液中IFN-γ分泌水平。比较哪种方法干预对免疫细胞功能的恢复影响更大。
结果：
1.干预前的T淋巴细胞(未经中药血浆干预也未与DCs共培养的T细胞)上CD4、CD28的表达水平,健康人组明显高于免疫耐受组和免疫清除组,而以免疫耐受组的患者表达最低,差异有统计学意义(p<0.05)。CD8、PD-1的表达健康人组明显低于免疫耐受组和免疫清除组,其中又以免疫耐受组的表达最高,差异有统计学意义(p<0.05)。CD3的检测结果健康人和免疫清除期组患者差异无显著意义(p>0.05),但耐受期组患者表达明显低下(p<0.05)。上清液中分泌的IFN-γ水平在健康人和免疫清除期组患者差异无显著意义(p>0.05),在耐受期组患者分泌水平明显低下(p<0.05)。
2.在免疫耐受组和免疫清除组的HBV慢性感染患者经补肾解毒(六味甘露中剂量)和健脾解毒(四君甘露小剂量)药物血浆干预后的DC与T细胞共培养,检测其T细胞表面分子的表达率。两种方法在一定程度上都能提高CD3、CD4、CD28的表达水平和上清液中分泌的IFN-γ水平,降低CD8、PD-1的表达水平。
结论：
1.慢性HBV感染免疫清除期和免疫耐受期患者存在T淋巴细胞功能的缺陷,以免疫耐受期患者细胞免疫功能是最低的。
2.通过补肾解毒、健脾解毒药物血浆在一定程度上能够促进HBV慢性感染患者树突状细胞的成熟,使得DCs激活T细胞的功能明显提高,促进T细胞表面分子的表达,并且补肾解毒法对慢性HBV感染免疫耐受期患者细胞免疫学功能的影响更为显著。
Objective:Using HBV chronic infection process different stages of the patients peripheral blood DCs and T lymphocytes of in vitro cultured cells model and traditional Chinese medicine pharmacology experiment method of plasma,comparisoning bushenjiedu and jianpijiedu which method can promote detoxification of DCs activation specific T lymphocytes, Which method is to improve the HBV infection after chronic immune cells function, immunological responses recovery, and the treatment of chronic hepatitis b main method, which for clinical improving the curative effect of TCM therapy for chronic hepatitis b provides an objective basis, treatment idea and research foundation.
Method:This study by extracting HBV chronic infection immune tolerance period and immune clearance phase16patients peripheral blood80ml. Another set8cases of healthy people as control group, To cultivate a separation of mature dendritic cells, in the process of cultivation using filling kidney detoxify and spleen detoxification compound Chinese medicine drugs respectively in plasma DCs intervention, and load HBV core protein, then with own T lymphocytes trained72hours, Application flow cytometry technical inspection T cells molecular CD3, surface, CD8, CD28CD4and PD-1expression, And ELISA test kit on T cells in the fluid of qing IFN-y secretion levels. comparisoning which kind of method is intervention on the immune cells function restore much more influence.
Results:
1.Before the intervention of the CD4+T lymphocytes, CD28+expression level, healthy human group was significantly higher than the immune tolerance group and immune clearance group, and group of patients with immune tolerance to express the minimum(p<0.05). CD8+, PD-1expression of healthy people obviously lower than immune tolerance group and immune clearance group, which the highest expression of immune tolerance(p<0.05), CD3+results of the health and immune clearance phase groups of patients, no significant difference (p>0.05), but the resistance stage patients with low obvious group expression(p<0.05). Clear liquid secretion on IFN-γ level in health and immune clearance phase groups of patients, no significant difference (p>0.05), in tolerance period groups of patients secretion level obviously low (p<0.05).
2.The immune tolerance group and immune clearance group of HBV chronic infection by the spleen and kidney patients with detoxification detoxification medicine after intervention of DC and plasma T cells of trained, testing the T cells surface of molecular expression rate. The two methods in a degree, can improve CD3+, CD4+, CD28+expression level and clear liquid on the secreted IFN-γ level, reduce the CD8+, PD-1expression level.
Conclusion:
1. Chronic HBV immune clearance phase and immune tolerance were present period T lymphocyte function of the defect, immune-tolerant phase in patients with cellular immune function is the lowest.
2. Through the filling kidney detoxification, invigorating spleendetoxification medicine in a certain extent plasma can promote chronic HBV infection were the mature dendritic cells, making the DCs activate t-cells function improved obviously, promote T cells the expression of molecular surface, and filling kidney detoxification method to chronic HBV immune tolerance of the influence of the patient with cells immunological function is more significant.
引文
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