壮药汗衣台药效物质基础及抗乙型肝炎病毒作用机制研究

Zhuang herb Hanyitai (Zhuang language) is also called “find the root of thousand miles”, it is the rattan of Tinospora (Tinospora crispa (L.) Miers) of Menispermaceae plants and scattered mainly in Yunnan and Guangxi provinces. It has the function of promoting blood circulation, clearing heat and toxin, anti-inflammation pain , lowering blood sugar and blood pressure, anti-proliferative effect and so on. the plant has been showed a good effect on chronic hepatitis B treatment in Guangxi HengXian and LongZhou Zhuang folk, but this effect has not been reported. Our previous studies have demonstrated for the first time that the the Hanyitai has the effect of anti-hepatitis B virus in vitro and in vivo..In this study, first of all, Hanyitai will be separated and purified and signed in order to gain monomer, then one monomer or more monomers combination will be taken as the research objectes. These experimentes will be carried out by fluorescence PCR, recombinant plasmid construction and liposome transfection, immunohistochemistry and Western blot, flow cytometry, ELISA, in order to detect HBVcccDNA copy number, HBV promoters activity, the transcription and expression of anti-viral proteins in cells infected, immune cells’ related cytokines release and receptors expression, The cytokines level associated with immune in serum and cytokine genes mRNA transcription level associated also with immune in spleen lymphocyte in chronic DHBV infection duck model. It is said that three aspects of this research including Hanyitai’s direct inhibition to HBV virus, indirect effect on antiviral protein in infected cells, immune function activation of immune cells and infected body. It will be revealed that the effective ingredients and the mechanism of the Hanyitai on anti-HBV, and provide a theoretical basis for a new anti-HBV drug.

汗衣台(壮语)别称千里找根，为防己科植物皱波青牛胆的藤茎，主要分布于云南和广西，具有通经活血，清热排毒，消炎止痛，降血糖，降血压，抗增殖等作用。广西横县龙州一带壮族民间流传其治疗慢乙肝有良效,文献未有记载，我们的前期研究首次证实其具有抗HBV活性。.本研究首先对汗衣台分离提取纯化鉴定其化合物单体，以单体及多单体配伍为研究对象，通过荧光定量PCR、重组质粒构建及脂质体转染、免疫组化及Western blot、流式细胞术、ELISA等方法，分别检测HBVcccDNA拷贝数、HBV启动子活性、感染细胞多种抗病毒蛋白基因转录和蛋白表达、免疫细胞多种免疫相关细胞因子释放及受体表达、慢性HBV感染鸭血清及脾细胞多种免疫相关细胞因子水平及基因mRNA转录情况。即从汗衣台对HBV病毒的直接抑制、影响感染细胞抗病毒蛋白产生、刺激免疫细胞及感染机体免疫功能活化三方面作用，了解汗衣台抗HBV有效成分及其作用机制

汗衣台(壮语)别称千里找根，为防己科植物皱波青牛胆的藤茎，主要分布于云南和广西，具有通经活血，清热排毒，消炎止痛，降血糖，降血压，抗增殖等作用。广西横县龙州一带壮族民间流传其治疗慢乙肝有良效,文献未有记载，我们的前期研究首次证实其水煎液具有抗HBV活性。为进一步研究其活性成分的作用，本研究首先对汗衣台分离提取纯化鉴定其化合物单体，以单体及多单体配伍为研究对象，通过荧光定量PCR、重组质粒构建及脂质体转染、免疫组化及Western blot、流式细胞术、ELISA等方法，分别检测HBVcccDNA拷贝数、HBV启动子活性、感染细胞多种抗病毒蛋白基因转录和蛋白表达、免疫细胞多种免疫相关细胞因子释放及受体表达、慢性HBV感染鸭血清及脾细胞多种免疫相关细胞因子水平及基因mRNA转录情况。即从汗衣台对HBV病毒的直接抑制、影响感染细胞抗病毒蛋白产生、刺激免疫细胞及感染机体免疫功能活化三方面作用，了解汗衣台抗HBV有效成分及其作用机制。实验结果表明，汗衣台有十多种有效成分，包括古伦宾、异古伦宾、非洲防己碱、掌叶防己碱、药根碱、蝙蝠葛碱、粉防己碱、轮环藤碱、木兰花碱、巴马亭红碱、千斤藤碱, 选择其中6种含量较多的有效成分做研究，发现汗衣台6种主要成分均具有不同程度的抗HBV作用，均可视为抗HBV的有效成分。6种有效成分不同程度抑制HBVDNA复制，对HBVcccDNA的抑制呈剂量依赖性; 选取在相同浓度下对HBsAg抑制率最高的非洲防己碱(83.9%)和抑制率最低的古伦宾(48.8%)相对比，研究两种成分对HBV启动子的作用，汗衣台抗HBV有效成分古伦宾、非洲防己碱对经典型和突变型的4种HBV启动子Cp，S1p，S2p，Xp均有不同程度的抑制作用，与干扰素和拉米夫定类似。四种药物对Xp尤其是突变型Xp有明显抑制作用，其中以干扰素最显著，提示汗衣台除了通过抑制启动子活性抗HBV以外，可能还通过抑制Xp启动子活性，继而下调X蛋白的表达，从而对HBV相关性肝癌的发生有预防作用。另含量较少的粉防己碱具有抗肿瘤药物增敏剂的作用，可以有效减少肿瘤细胞膜上药物泵蛋白的表达而对肿瘤细胞耐药具有逆转作用。