RNA干扰技术对柯萨奇B组3型病毒的抑制作用研究

发布时间：2018-11-27 09:59

【摘要】：第一部分：CVB_3-VP_1 siRNA对CVB_3体外复制的抑制作用目的：RNA干扰(RNA interference，RNAi)是近年发现的研究生物体基因表达、调控与功能的一项崭新技术，是由小干扰RNA引起的生物细胞内同源基因的特异性沉默(silencing)现象。利用RNAi技术研究对CVB_3的复制的抑制作用，观察柯萨奇B组病毒3型衣壳蛋白1(CVB_3-VP_1)特征性小干扰RNA(small interferingRNA，siRNA)对CVB_3复制及VP_1蛋白表达的抑制作用，探索抗CVB病毒感染的基因治疗新的方法。为CVB感染性疾病的治疗提供理论基础。方法：根据CVB_3 VP_1基因的序列及其结构特征，，设计并用化学方法合成4对siRNA。以CVB_3感染HeLa细胞，实验病毒用量为100μL内含100TCID_(50)病毒液／孔，18小时后通过脂质体介导以siRNA转染HeLa细胞，siRNA用量为3μL(60pmol)。七组HeLa细胞分别为转染CVB_3 VP_1-1 siRNA组，转染CVB_3 VP_1-2 siRNA组，转染CVB_3 VP_1-3 siRNA组，转染CVB_3 VP_1-4 siRNA组，转染no silencing-siRNA组，阳性对照组，空白对照组。48h后观察细胞病变的程度、TCID_(50)法测定病毒滴度、用免疫荧光法测定CVB_3-VP_1蛋白表达，RT-PCR法检测CVB_3-RNA的水平。结果：(1)CVB_3 RNA检测 siRNA组中VP_1-1、VP_1-2和阴性对照组无扩增条带，而阳性对照组与非特异siRNA及VP_1-3、VP_1-4组均可见827bp靶基因的条带。(2)免疫荧光法测定CVB3-VP1蛋白表达半定量分析表明，感染病毒后再转染VP_1-3、VP_1-4组及非特异siRNA与阳性对照组相比较无统计学意义(P＞0.05)；而转染VP_1-1，VP_1-2 siRNA与阳性对照组比较差异有显著性(P＜0.05)。(3)HeLa细胞病理变化在相差显微镜下观察到在VP_1-3和VP_1-4组大多数细胞肿胀、变圆、卷缩变形及空斑形成，而转染VP_1-1和VP_1-2组的细胞则只有少数细胞死亡。提示VP_1-1和VP_1-2组对CVB_3感染的HeLa细病变程度较轻，具有明显的保护作用，而VP_1-3和VP_1-4的保护作用不明显。(4)病毒滴度测定显示，VP_1-1和VP_1-2 siRNA组病毒滴度为0，而VP_1-3、VP_1-4
[Abstract]:Part I: inhibitory effect of CVB_3-VP_1 siRNA on CVB_3 replication in Vitro objective: RNA interference (RNA interference,RNAi) is a recent discovery of gene expression in organisms. A new technique for regulation and function is the phenomenon of specific silencing of homologous genes in biological cells caused by small interfering RNA. The inhibition of CVB_3 replication was studied by RNAi technique, and the characteristic small interference RNA (small interferingRNA, of coxsackie B virus type 3 capsid protein 1 (CVB_3-VP_1) was observed. SiRNA) inhibits CVB_3 replication and VP_1 protein expression, and explores a new gene therapy method against CVB virus infection. To provide a theoretical basis for the treatment of CVB infectious diseases. Methods: according to the sequence and structure of CVB_3 VP_1 gene, four pairs of siRNA. were designed and synthesized by chemical method. HeLa cells were infected with CVB_3. The amount of the virus was 100 渭 L containing 100 TCID _ (50) virus / well. After 18 hours, the HeLa cells were transfected with siRNA via liposome and the dosage of siRNA was 3 渭 L (60pmol). The seven groups of HeLa cells were transfected with CVB_3 VP_1-1 siRNA group, CVB_3 VP_1-2 siRNA group, CVB_3 VP_1-3 siRNA group and CVB_3 VP_1-4 siRNA group, respectively. The degree of cytopathic changes was observed 48 hours after transfection into no silencing-siRNA group, positive control group and blank control group. The viral titer was measured by TCID_ (50) method, and the expression of CVB_3-VP_1 protein was detected by immunofluorescence assay. The level of CVB_3-RNA was detected by RT-PCR. Results: (1) there were no amplified bands in VP_1-1,VP_1-2 and negative control group in siRNA group by CVB_3 RNA detection, but there were no amplified bands in positive control group and non-specific siRNA and VP_1-3, in positive control group. 827bp target gene bands were found in VP_1-4 group. (2) Semi-quantitative analysis of CVB3-VP1 protein expression by immunofluorescence assay showed that VP_1-3, was transfected after infection with the virus. There was no significant difference in siRNA between VP_1-4 group and positive control group (P > 0. 05). VP_1-1, transfection There was a significant difference between VP_1-2 siRNA and the positive control group (P < 0. 05). (3). The pathological changes of HeLa cells were observed under phase contrast microscope. Most of the cells in VP_1-3 and VP_1-4 groups were swollen and round. Only a few of the cells transfected with VP_1-1 and VP_1-2 died. It was suggested that VP_1-1 and VP_1-2 groups had a mild and obvious protective effect on HeLa with CVB_3 infection, while VP_1-3 and VP_1-4 had no obvious protective effects. (4) the virus titer assay showed that there was no significant protective effect on HeLa infection. The titers of VP_1-1 and VP_1-2 siRNA groups were 0 and VP_1-3,VP_1-4.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R373

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