【摘要】：第一部分：CVB_3-VP_1 siRNA對CVB_3體外復(fù)制的抑制作用 目的：RNA干擾(RNA interference，RNAi)是近年發(fā)現(xiàn)的研究生物體基因表達、調(diào)控與功能的一項嶄新技術(shù)，是由小干擾RNA引起的生物細胞內(nèi)同源基因的特異性沉默(silencing)現(xiàn)象。利用RNAi技術(shù)研究對CVB_3的復(fù)制的抑制作用，觀察柯薩奇B組病毒3型衣殼蛋白1(CVB_3-VP_1)特征性小干擾RNA(small interferingRNA，siRNA)對CVB_3復(fù)制及VP_1蛋白表達的抑制作用，探索抗CVB病毒感染的基因治療新的方法。為CVB感染性疾病的治療提供理論基礎(chǔ)。 方法：根據(jù)CVB_3 VP_1基因的序列及其結(jié)構(gòu)特征，，設(shè)計并用化學方法合成4對siRNA。以CVB_3感染HeLa細胞，實驗病毒用量為100μL內(nèi)含100TCID_(50)病毒液／孔，18小時后通過脂質(zhì)體介導(dǎo)以siRNA轉(zhuǎn)染HeLa細胞，siRNA用量為3μL(60pmol)。七組HeLa細胞分別為轉(zhuǎn)染CVB_3 VP_1-1 siRNA組，轉(zhuǎn)染CVB_3 VP_1-2 siRNA組，轉(zhuǎn)染CVB_3 VP_1-3 siRNA組，轉(zhuǎn)染CVB_3 VP_1-4 siRNA組，轉(zhuǎn)染no silencing-siRNA組，陽性對照組，空白對照組。48h后觀察細胞病變的程度、TCID_(50)法測定病毒滴度、用免疫熒光法測定CVB_3-VP_1蛋白表達，RT-PCR法檢測CVB_3-RNA的水平。 結(jié)果：(1)CVB_3 RNA檢測 siRNA組中VP_1-1、VP_1-2和陰性對照組無擴增條帶，而陽性對照組與非特異siRNA及VP_1-3、VP_1-4組均可見827bp靶基因的條帶。(2)免疫熒光法測定CVB3-VP1蛋白表達半定量分析表明，感染病毒后再轉(zhuǎn)染VP_1-3、VP_1-4組及非特異siRNA與陽性對照組相比較無統(tǒng)計學意義(P＞0.05)；而轉(zhuǎn)染VP_1-1，VP_1-2 siRNA與陽性對照組比較差異有顯著性(P＜0.05)。(3)HeLa細胞病理變化 在相差顯微鏡下觀察到在VP_1-3和VP_1-4組大多數(shù)細胞腫脹、變圓、卷縮變形及空斑形成，而轉(zhuǎn)染VP_1-1和VP_1-2組的細胞則只有少數(shù)細胞死亡。提示VP_1-1和VP_1-2組對CVB_3感染的HeLa細病變程度較輕，具有明顯的保護作用，而VP_1-3和VP_1-4的保護作用不明顯。(4)病毒滴度測定顯示，VP_1-1和VP_1-2 siRNA組病毒滴度為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.
【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R373

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