【作者】 朱欣；

【导师】 李闻文；

【作者基本信息】 中南大学 ， 临床检验诊断学， 2008， 硕士

【摘要】 目的:探讨麻黄水提液在体外抗呼吸道合胞病毒（RSV）的作用及其作用机制,为治疗呼吸道合胞病毒感染寻找新型药物。方法:1、将中药麻黄加水熬制,采用负压抽滤后合并滤液,用减压蒸发的方法浓缩制成水提液。2、采用体外细胞培养技术,通过目测法和MTT法检测麻黄水提液对Hela细胞的毒性。3、以利巴韦林为阳性对照,对麻黄水提液进行体外抗RSV的药效学检测。用空斑减数实验测得麻黄和利巴韦林抑制RSV的半数有效剂量。用细胞病变抑制实验检测麻黄水提液对RSV的吸附与穿入的抑制作用以及其对RSV的直接抑制作用。用空斑减数实验检测麻黄水提液在不同浓度不同时间对RSV的抑制作用。结果:1、麻黄水提液对Hela细胞的半数中毒浓度(CC₅₀)目测法为12.58mg/ml,MTT法结果为12.21 mg/ml,利巴韦林对Hela细胞的CC₅₀目测法为0.42 mg/ml,MTT法为0.44 mg/ml。麻黄水提液对RSV的半数有效剂量(EC₅₀)为3.74mg/ml,选择系数（SI）为3.26,利巴韦林EC₅₀为35.66μg/ml,SI为12.34。2、麻黄水提液和利巴韦林对RSV吸附到Hela细胞上的过程和穿入细胞膜的过程均有明显的抑制作用（P＜0.01）;麻黄水提液对RSV还有直接抑制作用（P＜0.05）;5.00mg/ml、4.00mg/ml、3.20 mg/ml、2.56 mg/ml麻黄水提液4个剂量组在RSV感染后8小时内给药均能抑制RSV（P＜0.05）;各浓度间,相同浓度不同时间给药对病毒的抑制均有差异（P＜0.05）,时间和浓度两个因素之间存在交互作用。浓度越高,用药时间越早,抑制RSV的作用越大;浓度越低,用药时间越晚,对RSV的抑制作用越小。利巴韦林在8小时内给药也对RSV有抑制作用。结论:1、5.00mg/ml麻黄水提液能阻止RSV对细胞的吸附,也能抑制RSV对细胞膜的穿透。2、麻黄水提液2.5mg/ml时就对RSV有直接抑制作用。3、麻黄水提液5.00mg/ml、4.00mg/ml、3.20 mg/ml、2.56 mg/ml4个剂量组在感染8小时内给药均能抑制RSV,而且对RSV的抑制作用有明显的量效、时相关系。4、麻黄水提液在体外具有明显的抑制RSV作用。更多还原

【Abstract】 Objective:1、In order to find the new drug for respiratory syncytial virus（RSV） infection treatment, we study the effect and mechanism of anti-respiratory syncytial virus of the water extract of Ephedra Sinica.Methods:1、We decoct the Chinese medicine Ephedra Sinica by adding water, then drainaging and filtering it under the negative pressure before we collect the filtrate. We concentrate the medicial solution and make it into Ephedra Sinica water extract using the method of vacuum evaporation.2、The cytotoxicity of Ephedra Sinica water extract was tested by MTT assay and visual observation using the method of cell culture in vitro.3、Antiviral activity of the Ephedra Sinica water extract was tested by the method of cell culture using ribavirin as positive control. The median effective concentrations (50% effective concentration, EC₅₀) of the Ephedra Sinica water extract and ribavirin were tested by the plaque reduction method. Using the CPE inhibition assay, the cytopathic effect of the drugs in the inhibition assay for virus absorption and penetration were tested, and the direct effect of it on RSV was also be determined. Antiviral activity of Ephedra Sinica at different concentrations in the time-course were tested by the plaque reduction method.Result: 1、The median cytotoxicity concentration(50% cytotoxicity concentration, CC₅₀) of Ephedra Sinica was 12.58 mg/ml and 12.21 mg/ml by visual assessment and MTT assay respectively. The median cytotoxicity concentration of ribavirin was 0.42 mg/ml by visual assessment and it was 0.44 mg/ml by MTT assay. Using the plaque reduction method, the median effective concentration(50% effective concentration, EC₅₀) of the extract of Ephedra Sinica was 3.74 mg/ml and the median effective concentration of the ribavirin was 35.66μg/ml. The selectivity index（SI） of Ephedra Sinica was 3.26 and the SI of ribavirin was 12.34.2、Ephedra Sinica and ribavirin could inhibit RSV adhering to the cells（P<0.01） , they could also inhibit RSV penetrating into the cells （P<0.05）; Ephedra Sinica also showed direct inhibition on RSV（P<0.05）; the groups of 5.00mg/ml、4.00mg/ml、3.20 mg/ml、2.56 mg/ml of Ephedra Sinica water extract showed obvious effect of inhibition on RSV when it was added in 8 hours after RSV infection（P<0.01）; ribavirin also showed obvious effect of inhibition on RSV when it was added in 8 hours after RSV infection（P<0.01）.3、The difference of the medication with different strength groups and the difference of medication at different times had statistical signifycance by Factorial Analysis. There was interaction between the time and the strength of medication. The higher the concentration, the earlier the administration time, more effects on virus inhibitation. The lower the concerntration, the later the treatment began, least effects on virus inhibitation.Conclusion:1、5.00mg/ml Ephedra Sinica water extract could inhibit RSV adhering to the cells, it also could inhibit RSV penetrating into the cells.2、Ephedra Sinica water extract at the concentration of 2.50mg/ml had direct inhibition on RSV.3、the groups of 5.00mg/ml、4.00mg/ml、3.20mg/ml、2.56 mg/ml of Ephedra Sinica water extract had anti-RSV effect when it was added in 8h after RSV infection. There were a dose-dependent response and phase realationship of Ephedra Sinica in inhibiting RSV.4、Ephedra Sinica water extract showed potent inhibition on RSV in vitro.更多还原