【作者】 韩英光；

【导师】 何清湖；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的：探讨中药龙胆泻肝配方颗粒对生殖器疱疹病毒增殖的抑制作用；通过研究龙胆泻肝配方颗粒对生殖器疱疹患者外周血树突状细胞表面成熟标志CD83、CD86表达的影响及对树突状细胞分泌白细胞介素-12水平的影响,探讨龙胆泻肝配方颗粒免疫作用机制的靶点。方法：1)采用细胞培养技术对龙胆泻肝配方颗粒水溶物体外抗HSV-2的药效实验进行研究。在增殖抑制法、感染阻断法及直接杀灭法3种作用方式下,不同浓度的药物对生殖器疱疹病毒增殖的影响。2)通过体外细胞培养的方法用龙胆泻肝配方颗粒水溶物对生殖器疱疹患者的树突状细胞进行作用,然后检测患者及健康者外周血DC的表面标志CD83、CD86的表达水平。3)用ELISA法检测正常人的对照组和生殖器疱疹患者组DC的培养上清液中的IL-12含量。结果：1)药物的细胞毒性测定：正常细胞对照的CPE均小于25％,龙胆泻肝配方颗粒及阿昔洛韦(ACV)对Vero细胞的最大无毒浓度(TC0)分别为0.16、0. lmg/ml.按Reed-Muench法算出龙胆泻肝配方颗粒、ACV对Vero细胞的TC50分别为5.631、0.457mg／ml。2)病毒感染性测定：将病毒原液行连续10倍递次稀释,进行病毒感染性测定,测得HSV-2 333病毒株的TCID50=10-4／0.1ml。3)药物对HSV-2所致CPE的抑制实验结果：三种不同药物加入方式下,实验所设病毒对照组均可见典型CPE,正常细胞对照组均无明显病变。4)MTT法药物抗HSV-2的药效：用MTT法测定正常细胞对照组及病毒对照组平均A490值分别为0.294±0.053、0.032±0.0065。方式Ⅰ加药,几乎各浓度中药实验组和西药对照组的平均A490值与病毒对照组比较,均有统计学差异P(<0.05或P<0.01)；方式Ⅱ加药,部分浓度中药实验组的平均A490值与病毒对照组比较,有统计学差异(P<0.05)；方式Ⅲ加药,几乎各浓度中药实验组的平均A490值与病毒对照组比较,均有统计学差异(P<0.05或P<0.01)；方式Ⅱ和Ⅲ加药,西药对照组的A490平均值与病毒对照组比较,均无统计学差异(P>0.05)。5)以药物浓度为横坐标(μg／ml)、病毒抑制率(IR％)为纵坐标绘出不同药物加入方式下的剂量效应关系图。方式IACV对HSV-2的抑制作用随浓度升高而增强,存在明显的量效关系；而龙胆泻肝颗粒量效应关系不甚明显。中药浓度达到40μg／ml时,其对HSV-2的抑制作用随浓度升高反而降低。方式Ⅱ中药浓度超过20μg／ml,其对HSV-2的抑制作用随浓度升高而增强.方式Ⅲ中药浓度达到80μg／ml时也出现IR随浓度升高而降低。6)正常人的空白对照组DC表面标志CD83、CD86的表达较低；而正常人中药对照组的DC的表面标志CD83、CD86的表达明显上调,与空白对照组比较有显著性差异(P<0.05)；生殖器疱疹患者对照组的DC的表面标志CD83、CD86的表达明显增高,与正常人空白对照组比较有显著差异(P<0.01)。生殖器疱疹患者中药作用组加入中药后的DC表面标志CD83、CD86与患者对照组比较表达下调,有显著性差异(P<0.01)7)正常人的空白对照组IL-12的含量为84.57ng／ml,生殖器疱疹患者对照组的含量与正常人的空白对照组比较明显升高,有显著性差异(P<0.01)；而在正常人的中药对照组的培养上清的IL-12的含量与正常人的空白对照组比较有所降低,但差异无统计学意义(P>0.05)。在生殖器疱疹患者中药组的培养上清的IL-12的含量与患者对照组比较明显下调,有显著性差异(P<0.01)。结论：1)浓度低于0.16mg／ml的龙胆泻肝配方颗粒对Vero细胞无毒,对Vero细胞非常安全。2)龙胆泻肝配方颗粒和阿昔洛韦均能不同程度地抑制Vero细胞内HSV-2的增殖,龙胆泻肝配方颗粒对HSV-2有直接杀灭作用,且对HSV-2的感染有一定的预防作用,而ACV无直接杀灭及预防作用。龙胆泻肝配方颗粒体外从多个作用环节较强地抑制HSV-2。3)ACV对HSV-2增殖抑制有明显的剂量一效应关系；龙胆泻肝配方颗粒对HSV-2感染阻断作用存在量效关系；但其对HSV-2增殖抑制及直接杀灭作无明显的剂量一效应关系。4)中药龙胆泻肝配方颗粒作用于正常人外周血树突状细胞后,细胞递呈抗原的能力明显增强,中药作用于生殖器疱疹患者的外周血树突状细胞后,细胞递呈抗原的能力受到一定的抑制,说明龙胆泻肝配方颗粒对外周血树突状细胞的抗原递呈功能起到一定调节作用。5)中药作用于外周血树突状细胞后,无论正常人还是患者,其树突状细胞的细胞因子IL-12的产生均强烈受抑制,且远远低于正常人水平,说明龙胆泻肝配方颗粒主要以细胞因子IL-12的产生为作用点来抑制Th1型的淋巴细胞的分化,以治疗生殖器疱疹患者。更多还原

【Abstract】 Objective To probe the inhibitory effect of Longdanxiegan Chinese traditional medicine Granule on replication of herpes simplex virus (HSV-2) in Vero cells in vitro. To explore the immunological function of Longdanxiegan Granule,through studying the Dendritic cells extracted from the patients’blood by cell culture method in vitro.Methods 1) methods as following:Method I, inhibiting virus multiplication-Confluent cell monolayers in 96-well plates were infected with HSV-2 333in medium. After adsorption at 37℃for 1.5 h, residual inoculum was replaced with 0.2ml of medium containing test drug.MethodⅡ, interdicting cells infection-Cell cultures were treated with the drugmedium for 24h,washed thrice with PBS,100 TCID50 of virus were addedto give 0.1ml per well and subsequently reincubated in drug-free medium.Method III, directly killing virus-Virus were incubated in drug medium for 24h at 37℃was added to give 0.2 ml per well.2) DC in peripheral blood in patient and healthy people were cultured for five days with medium which had GM-CSF and IL-4.Flow cytometry was used to analyze surface marker of CD83 and CD86 of DC.3) the content of IL-12 in cell culture supernatant was detected by ELISA.Result 1) The results shew that the TCo and the TC50 for Longdanxiegan were 0.16 and 5.631 mg/ml, respectively. Corresponding values for ACV were 0.1 and 0.457 mg/ml, respectively.2) The Infected capability of diluted stocks of HSV-2333 strains was measured by CPE method. The results shew that the TCID50 of HSV-2 for Vero cell was 10-4/0.1ml.3) By different drug addition methods, the typical cytopathic effects in Vero cells were observed in all parallel virus controlled groups, while normal Vero cellsgroups were almost negative. By drug addition I method, the CPEs were restrained at different degrees in Longdanxiegan and ACV groups; the CPEs were only inhibited at different degrees in Longdanxiegan group under drug addition method II and 111, while the cytopathic effects were clearly visible in ACV group.4) Measured by MTT colorimetry, the mean value of A490 for the parallel virus controlled groups and normal Vero cells groups were 0.294±0.053、0.032±0.0065, respectively. By drug addition I method, compared with the values of A490 of virus controlled groups,the values of A490 of Longdanxiegan and ACV in all concentrations were almost statistically significant (P<0.05 or P<0.01).By drug addition II method, The part values of Longdanxiegan group were statistically differences compared with that of virus controlled group (P<0.05). By the last drug addition method, only the values of Longdanxiegan group was statistically significant compared with that of the virus controlled group (p<0.01).The values of acyclovir group were not statistically differences (P>0.05) under drug addition method II andⅢ. 5) The charts of dose-effect by different drug addition methods were drawn, serving drug concentrations and IR% as abscissa and y-axis respectively. Under drug addition method I, the inhibitory effects of ACV for HSV-2 became stronger with the increase of concentration, showing clear dose-effect connection. At beginning, the inhibitory effects of Longdanxiegan became stronger with the increase of concentration. After exceeding 40pg/ml, it’s inhibitory effects declined with the increase of concentration. By drug addition II method, the inhibitory rates of Longdanxiegan for HSV-2 became stronger with the increase of concentration exceeding 20μg/ml. By the last drug addition method, the IR% of Longdanxiegan became weaker with the increase of concentration exceeding 80μg/ml too.6) The expression of CD83 and CD86 on DCs were higher in patients than that in healthy controls(P<0.05). After adding drug, the expression of these molecules were regulated, there were no significant differences between patients and controls(P>0.05).7) The production of IL-12 was increased obviously in control group of patient (p<0.05). After adding drug, the production of IL-12 was decreased evidently(p<0.01).Conclusion 1) Under the concentration of 0.16 mg/ml, Longdanxiegan were innocuity to Vero cells, Longdanxiegan was very safe to Vero cells.2) Multiplication of HSV-2 in Vero cells were inhibited at different degrees in Longdanxiegan and ACV groups. Only Longdanxiegan shew the activity of directly killing virus and guarding against Vero cells’infection by HSV-2 at different degree. Longdanxiegan had an activity for stronginhibition of HSV-2 at several effect points in vitro.3) The inhibitoryeffects of ACV for multiplication of HSV-2 shew clear dose-effect connection.The effects of Longdanxiegan for guarding against Vero cells’infection became stronger with the increase of concentration,but Longdanxiegan didn’t show clear dose-effect connection in restraining multiplication of HSV-2 and directly killing virus.4) The compound traditional Chinese medicine has the function of immunoregulation on DC, it can make DC of healthy people have potent antigen presenting capabilities, which also can inhibit antigen presenting capabilities of DC in patient.5) The production of IL-12 was inhibited in patient and healthy subjects by compound traditional Chinese medicine,so the immune response of Thl is inhibited by compound traditional Chinese medicine,it could make the disease psoriasis vulgaris more better.更多还原