【作者】 梅少林；

【导师】 常雅萍；

【作者基本信息】 吉林大学 ， 免疫学， 2005， 硕士

【摘要】 为了研究单纯疱疹病毒性脑炎(HSE)的免疫损伤的致病机制以及检测人参皂甙Rg3 对单纯疱疹病毒性脑炎的治疗作用,采用腹腔注射单纯疱疹病毒1 型(HSV-1)建立HSE 小鼠模型,通过观察症状和体征、脑匀浆病毒滴定、感染脑组织进行HE 染色、病理切片观察形态学改变确定模型的成立。通过HSE 小鼠脑组织匀浆一氧化氮(NO)的含量测定,探讨氧化损伤因素在HSE 发病中促进神经组织损害的作用。分别在细胞水平和动物水平检测了Rg3 的免疫调节活性。观察了HSV-1 体外对T 细胞增殖和Th1 型细胞因子分泌的影响。结果显示:实验浓度Rg3 对正常传代Vero 细胞及小鼠脾细胞增殖无毒性作用,并具有体外抗单纯疱疹病毒活性。NO 可加重HSV 所致脑组织损伤。Rg3 在小鼠能促进淋巴细胞转化,增强NK 细胞毒活性,提高Th1 型细胞因子水平。HSV-1 体外能促进小鼠T 细胞增殖和Th1 型细胞因子分泌。结论:HSV-1 所致的脑炎可能与局部T 淋巴细胞浸润引起的迟发性超敏反应有关。多种细胞因子参与调节HSV-1 所致炎症反应。NO 介导了HSE 的氧化损伤。Rg3 体外具有抗HSV-1 效应,体内外均具有免疫增强效应,只有合理用于HSE疾病进程的特定阶段才能调节机体的免疫状态,改变与机体不利的免疫病理损伤。更多还原

【Abstract】 As for the viral infection, foreign researches have been devoting theirenergy to develop antiviral medicine and vaccine. However, due to thespecificity of viral structures and entry sites and their large variability, theantiviral medicine and vaccine cannot keep up with the change of theepidemics. Traditional Chinese medicine has a long history of combatingwith epidemics for several millenniums. Research of the past few decadesshowed that the comparative advantages of Chinese medicines in preventingand treating viral infection are as follows. (1)They have a wide spectrumeffect of killing or inhibiting the viruses. (2)They can initiate thepreventative ability of organism and regulate the immunological function.(3)They are more safe and do not have obvious poisonous or side effect. Thedisadvantage of dysbacteriosis and double infection is very small. Thisexperiment investigated the antiviral effect of ginsenoside Rg3 on herpessimplex encephalitis (HSE) and its immunoregulative activity.This research adopted MTT colorimetry to detect the effect of Rg3 onthe proliferation of Vero cells. The results showed that TD0 of Rg3 is25ug/ml, whether incubating Rg3 and cells together or adding Rg3 afteroverlaying of the cells. Then the in vitro antiherpesvirus activity of Rg3 wasobserved from 3 aspects: (1)inhibiting the virus directly; (2)obstructingabsorption; (3)inhibiting replication. The result showed that when theconcentration of Rg3 is between 1.56 and 25ug/ml, it could significantlysuppress HSV-1 by adding it and virus together to the single cell layer. Whenadding the virus after the interaction of the medicine and cells, 25ug/ml Rg3has significant antiviral effect. For adding the medicine after attachment ofthe virus, the concentration of 3.13~25ug/ml can inhibit HSV-1 but have nostatistical significance. To investigate the effect of HSV-1 on the proliferation of Th1lymphocytes and the production of Th1 type cytokines, MTT colorimetrywas used to determine Th1 lymphocyte proliferation. In vitro L929 killingmethod was used to detect TNF-α, lymphoblast proliferation analysis wasused to detect IL-2, and IFN–γwas examined by cytopathic inhibitingmethod. Results: A certain titre of HSV-1 stimulated lymphocytetransformation significantly. The secretion of TNF-αand IL-2 increasedsignificantly. The level of IFN–γonly increased a little. Conclusion: HSV-1can activate the proliferation of Th1 lymphocytes and increase theproduction of Th1 type cytokines. It suggested that HSV-1 infection canstrengthen immune response. Excessive reponse may contribute to theimmunopathological damage to the organisms. HSE animal model was established by injecting intraperitoneally thevirus of 10 times serial dilution was 10-5 and the concentration to establishmodels should be 100 LD50. Treated the brain tissue by HE staining andobserved the morphological changes. Prepared brain tissue homogenate totitrate the virus and detect NO content. The mice showed symptoms andsigns after 3 or 4 days after inoculating the virus. The main symptoms were:decreased appetite, decreased body weight, tussled fur, tic, quiver, turningaround, hunch, lethargy, hemiplegia, paralyzing and die. The pathologicalexamination reported that there were congestion and edema in the braintissue. The necrosis of the neurons, softening foci, the lymphocytessurrounding the vessels, and glial nodes could also be seen. The detection ofNO in brain homogenate needed quantifying the content of protein first. Thecontent of NO was higher after viral infection compared to the normal group.The content was higher on day 3 than on day 5, but has no significantdifference compare to the normal group. The level of NO of virus group onday 5 was significantly different from that of normal group and hadstatistical meaning (p<0.05). It suggested that NO was a key mediator in thesecondary tissue damage after viral infection. Calculate the survival rate andextended survival rate of each group and the results showed that Rg3 hadprotective effect on HSE mice. The influence on the splenocytes proliferation was detected todemostrate in vitro immunoregulation of Rg3. Then two concentration ofeffective dose under TD0 was chosen to induce the supernatant ofsplenocytes and detect the secretion of Th1 type cytokines. The activity ofIL-2 and IFN-γin the supernatant induced by 6.25ug/ml, 3.13ug/mlRg3wassignificantly enhanced compared to control group (p<0.05). The in vivo immunoregulative effect was carried out by injectintraperitoneally with Rg3 and evaluated the following immunologicalindices: spleen index, lymphocyte transformation test, NK cytotoxicity, andthe level of Th1 type cytokines. The results showed that Rg3 couldsignificantly enhance the lymphocyte transformation function and improvethe content of cytokine. Spleen index and NK cytotoxicity also increased butthey did not have statistical meaning. Conclusion: Rg3 of certain concentrations had no poisonous effect onnormal cell lines and mouse splenocytes. It had in vitro anti-HSV activity.The experiment established mouse model of HSE successfully. The braindamage of HSE may correlate with the poisonous effect of Th1 cytokinesand NO on the neurons. Rg3 had protective effect on HSE mouse model. The更多还原