【作者】 唐颖蕾；

【导师】 夏雪山； 魏大巧；

【作者基本信息】 昆明理工大学 ， 微生物学， 2010， 硕士

【摘要】 丙型肝炎病毒(Hepatitis C virus,HCV)属于黄病毒科,肝炎病毒属,有包膜单股正链RNA病毒,基因组全长约9.6 kb。据估计,全世界约有1.7亿人感染HCV,感染人数不断增加,危害日益严重。HCV—经感染,约80%的病例会建立慢性感染,其中20%慢性感染者最终会发展成为肝硬化、肝癌。目前对丙型肝炎的治疗,主要采用聚乙二醇化干扰素(Peg-IFN)和利巴韦林(Ribavirin,RBV)联合疗法,但至少有约50%患者不能产生持续病毒学应答(SVR),1型HCV感染患者SVR率更低。同时,联合疗法还存在服用吸收率低、毒副作用大和成本偏高等缺点,因此迫切需要建立更为完善的药物筛选方法,筛选新的抗HCV药物。本研究在引进了2a型嵌合病毒(J6/JFH1)、Huh 7.5.1细胞HCV细胞培养体系(HCV cell culture,HCVcc)的基础上,经细胞培养条件优化和细胞成活率检测,确定了Huh7.5.1细胞的生长曲线。9×104cells/ml接种细胞密度情况下,经过潜伏期后,细胞2-6天进入大量增长的指数生长期,第3天细胞生长至可用作药物筛选的最佳密度。经过对不同感染复数的病毒在细胞中的增殖特性分析,发现病毒株感染细胞后3-7天,为生长的指数增长期,第3天病毒载量为6.5×106IU/ml,为病毒载量检测的最佳时间；病毒株对细胞的最适感染复数为1。在建立了检测药物细胞毒性MTT方法,和荧光定量PCR病毒载量检测方法的基础上,以干扰素α-1b(IFNα-1b)和利巴韦林(RBV)为阳性对照,建立了抗HCV药物筛选体系。结果显示：IFNα-1b无实际细胞毒性,半数有效浓度(EC50)值为1.452IU/ml;利巴韦林半数细胞抑制生长浓度(ICso)值为20.14μg/ml,半数有效浓度(ECso)值为2.553μg/ml,选择指数(SI)值为7.889。分别对IFNα-1b和RBV不同给药方式,不同时间的抗病毒效果检测,发现IFNa-1b预处理细胞18h后加入病毒,RBV在病毒同时感染同时加入,可达到最佳抗病毒效果,二者联合用药的抗病毒效果明显高于单独使用。用已建立的抗HCV药物筛选方法,初步对15种化合物进行抗病毒效果评价,发现其中6种化合物对HCV RNA的复制有明显抑制活性,分别为：shm-7(IC50,1.089μg/ml；EC50,0.025μg/ml；SI,44).LDX22(IC50,5.752μg/ml；EC50,0.180μg/ml；SI,32).LDX28a(IC50,20.390μg/ml；EC50,0.672μg/ml；SI,30).ZDH7(IC50, 32.640μg/ml；EC50,0.561μg/ml；SI,58).ZDH22(IC50,5.089μg/ml；EC50,0.260μg/ml；SI,20)、ZDH26(IC5o,14.870μg/ml；EC50,0.127μg/ml；SI,116)。其中LDX28a和ZDH7,无明显细胞毒性。其它9种药物(shm-4、shm-9a、shm-14、LDX24、ZCS3、ZCS4、ZCS11、ZCS17、ZDH17)的选择指数明显低于对照药物(RBV)。综上所述,本研究在建立的HCV细胞培养体系的基础上,以目前标准治疗药物干扰素和利巴韦林为阳性对照,建立了抗HCV药物筛选方法,初步应用于抗HCV药物筛选,从15种备选药物中发现了6种化合物具有一定的抗病毒活性。所建立的方法可以准确、快速的进行抗HCV药物的筛选,为充分利用云南特有药物资源开发抗病毒药物搭建了必要的技术平台。更多还原

【Abstract】 Hepatitis C virus (HCV), a small and enveloped RNA virus, was classified to Hepacivirus genus of Flaviviridae family. Its genome was a single- stranded positive-sense RNA of 9.6 kb length. It was estimated that 170 million people was infected with HCV worldwide. The increasing of infection case indicated the more severe prevalence. Once the occurrence of HCV infection, about 80% infected individuals will progressed to chronic disease. Among them, about 20% ended with liver failure and hepatocellular carcinoma. The current standard therapy was pegylated interferon-αand ribavirin combined treatment. However, less than 50% patients was shown with a sustained viral response (SVR), the effection of treatment is much poor in patients with HCV lb strains infection. In addition, the low effection rate, the obvious side effects and high cost had hampered its clinical application. It was urgent to develop the new treatment using the more convincing anti-HCV drug screening system.The successfully established 2a chimeric strains (J6/JFH1) HCV cell culture (Huh 7.5.1) system (HCVcc) had been introduced into our lab. With optimization of cell culture condition and analyze on Huh 7.5.1 cell growth curve, it was found that the cell grown exponentially between 2-6 culture days with started cell density of 9×104 cells/ml. And, the cultured No.3 day could be taken as the test time of cell toxicity. With the different infection multiplicity of infection and followd virus proliferation assay, the exponential growth phase was from 2 to 6 day after virus inoculation. The best determine time was No. 3 day with viral load of 6.5×106 IU/ml. The optimal multiplicity of infection of HCV strains was 1. Furthermore, IFNa-lb and Ribavirin was used as positive control to established MTT cytotoxic assay and real-time PCR viral load quantitaion method to evaluate the antiviral effect of drugs. It was shown that IFNa-lb have no obvious cell toxicity, which half effective concentration value was 1.452 IU/ml. For Ribavirin, half of the cell growth inhibition concentration (IC50) value was determined as 20.14μg/ml, whose half effective concentration (EC50) values was 2.553μg/ml, the calculated selective index (SI) value was 7.889. In parallel, IFNa-lb was shown no obvious cyto-toxicity with EC50 of 1.452 IU/ml. The different drug delivery time and patterns were performed furtherly, the 18 hours after virus inoculation and concurrent of inoculation was shown as the best start interrupte time for IFNa-lb and RBV respectively. The inhibite effect of combinate treatment was better than single using of IFNa-lb or Ribavirin.With the established screening system, the anti-HCV effects of 15 candidates were tested. Among them, the obvious inhibition effects of 6 compounds were proved. It was listed as shm-7 (IC50:1.089μg/ml, EC50:0.025μg/ml, SI,44), LDX22 (IC50:5.752μg/ml, EC50:0.180μg/ml, SI:32), LDX28a (IC50:20.390μg/ml, EC50:0.672μg/ml, SI:30), ZDH7(IC50:32.640μg/ml, EC50:0.561μg/ml, SI:58), ZDH22 (IC50:5.089μg/ml, EC50: 0.260μg/ml, SI:20) and ZDH26 (IC50:14.870μg/ml, EC50:0.127μg/ml, SI:116). LDX28a and ZDH7 hadn’t any cytotoxity. It was notable that selective index of ZDH26 was up to 116, which was much higher than positive control RBV. The anti-HCV effects of remained 9 compounds (shm-4, shm-9a, shm-14, LDX24, ZCS3, ZCS4, ZCS11, ZCS17 and ZDH17) haven’t shown here.In summary, based on the introduced HCV cell culture system, the anti-HCV drug screening system had been established with interferon and ribavirin as a positive control. Using this menthod, the anti-HCV effects of 6 compounds from 15 candidates was determined. This screening system will be taken as a useful tool to discover anti-HCV compound from the prolific drug resource of Yunnan. The promotion of this technique platform on drug industry could be expected.更多还原