龙胆泻肝配方颗粒对复发生殖器疱疹豚鼠Toll受体信号通路的影响

To explore the therapeutic mechanism of Longdanxiegan Formula Granule against recurrent genital herpes（RGH） on molecular biology level, through studying the femal guinea-pigs of RGH. To explore the immunologic mechanism of Longdanxiegan Formula Granule in the process of natural and acquired immune responses, through studying the Toll-like receptor signaling transdution pathway roundly on antigen presenting cell..We can make the model of female guinea-pig genital herpes(GH) by inoculating HSV-2 in vagina，then treat the guinea-pig GH with the deferent dose Longdanxiegan Formula Granule and Acyclovir respectively.20 days later,we made the model of guinea-pig RGH by inducing them to recur through peritoneal injection of Cytoxan(CTX)and local irradiation of ultraviolet(UV)．.We randomly divide the 480 animal models of female guinea-pig with RGH into 6 groups completely,a treatment group with low dose Longdanxiegan Formula Granule,a treatment group with middle dose Longdanxiegan Formula Granule,a treatment group with high dose Longdanxiegan Formula Granule,a control group with Aciclovir,another control group with saline water and a normal group. To assess the lesion changes in vulva and vagina of guinea pigs before and after the treatment..Peripheral blood mononuclear cells (PBMC) can be separated from heparinized venous blood by stimulating factor GM-CSF、IL-4、TNF-α. The Dendritic cells can be harvested for phenotypic analysis by flow cytometry.Then some phenotype changes such as CD11c、CD80、CD83、CD86、HLA-DR and HLA-ABC of DCs will be observed. RNA of the dendritic cells can be extracted by Trizol. The levels of mRNA for TLR1～TLR10 will be examined by fluorescent quantitative RT-PCR. And the levels of mRNA for MyD88 and NF-kB will be examined by RT-PCR.Then the expressions of IL-6、IL-8、IL-10、IL-12、IFN-ɑ、IFN-β、IFN-γ and TNF-ɑ in peripheral blood will be detected before and after treatment by ELISA..Furthermore,to randomly take 40 guinea-pigs every group and cut the lesion in vulva the second day after the treatment.Then density gradient centrifugation and magnetic cell sorting can be combined simultaneously to separate and purify the Langerhans cells from epidermal cell suspension. Then some phenotype changes such as CD1a、CD45 and MHC-Ⅱ of LCs will be observed.The levels of mRNA for TLR1～TLR10 will be examined by fluorescent quantitative RT-PCR.The levels of mRNA for MyD88 and NF-kB will be examined by RT-PCR also.

选用清热解毒中药龙胆泻肝配方颗粒，通过对复发性生殖器疱疹豚鼠的实验研究，来明确其抗HSV病毒的作用，系统地研究其对抗原递呈细胞表面Toll受体信号转导途径的作用机制。从以下三方面进行研究：龙胆泻肝配方颗粒对复发性生殖器疱疹豚鼠的疗效评价；龙胆泻肝配方颗粒对复发性生殖器疱疹豚鼠外周血树突状细胞TLR1～TLR10mRNA表达情况的影响，及对Toll样受体下游MyD88、NF-kB蛋白表达的影响，由Toll样受体所介导的细胞因子IL-6、IL-8、IL-10、IL-12、IFN-ɑ、IFN-β、IFN-γ、TNF-ɑ的变化情况；取豚鼠皮损处皮肤，分析其对表皮朗格汉斯细胞TLR1～TLR10mRNA表达情况，MyD88、NF-kB蛋白表达的影响。针对其抗病毒的作用机制，从免疫学和分子生物学的角度，提出明确的作用靶点，进一步阐明中药在激发固有免疫和获得性免疫反应中的作用机制及其分子基础。

选用清热解毒中药龙胆泻肝配方颗粒，通过对复发性生殖器疱疹豚鼠的实验研究，来明确其抗HSV病毒的作用，系统地研究其对抗原递呈细胞表面Toll受体信号转导途径的作用机制。从以下三方面进行研究：龙胆泻肝配方颗粒对复发性生殖器疱疹豚鼠的疗效评价；龙胆泻肝配方颗粒对复发性生殖器疱疹豚鼠外周血树突状细胞TLR1～TLR10mRNA表达情况的影响，及对Toll样受体下游MyD88、NF-kB蛋白表达的影响，由Toll样受体所介导的细胞因子IL-6、IL-8、IL-10、IL-12、IFN-ɑ、IFN-β、IFN-γ、TNF-ɑ的变化情况；取豚鼠皮损处皮肤，分析其对表皮朗格汉斯细胞TLR1～TLR10mRNA表达情况，MyD88、NF-kB蛋白表达的影响。针对其抗病毒的作用机制，从免疫学和分子生物学的角度，提出明确的作用靶点，进一步阐明中药在激发固有免疫和获得性免疫反应中的作用机制及其分子基础。