CXCR4-siRNA重组腺病毒载体的构建及其沉默CXCR4基因的体外研究

发布时间：2018-08-22 20:33

【摘要】：第一部分应用AdMax载体系统构建CXCR4-siRNA重组腺病毒载体目的：应用AdMax载体系统构建CXCR4-siRNA重组腺病毒载体，为研究其对人卵巢癌细胞株SKOV3的CXCR4基因表达的影响奠定基础。方法：将CXCR4基因的小干扰片段克隆至腺病毒穿梭质粒PAVsi1.1，通过PCR和测序筛选阳性克隆，将重组穿梭质粒、腺病毒辅助包装质粒、3Cre共转染HEK293细胞，包装产生CXCR4-siRNA重组腺病毒载体。在HEK293细胞中大量包装扩增病毒，收集病毒液，测定病毒滴度。结果：PCR和测序结果均提示CXCR4基因的小干扰片段成功构建至腺病毒穿梭质粒，通过HEK293反复包装扩增后，在荧光显微镜下可观察到HEK293细胞中绿色荧光蛋白的大量表达，病毒滴度为6×108PFU/ml。结论：AdMax载体系统可成功构建CXCR4-siRNA重组腺病毒载体。第二部分CXCR4-siRNA重组腺病毒载体沉默人卵巢癌细胞株CXCR4基因表达的研究目的：研究CXCR4-siRNA重组腺病毒载体对人卵巢癌细胞株SKOV3的CXCR4基因表达的影响。方法：将收集的大量病毒液转染人卵巢癌细胞株SKOV3。细胞分为三组：siRNA组，转染空病毒载体的阴性对照组，，细胞不做任何处理的空白对照组。通过PCR、Western blot、免疫组化检测CXCR4基因沉默效果。结果：病毒转染至SKOV3细胞后，在荧光显微镜下可观察到SKOV3细胞中绿色荧光蛋白的大量表达，重组腺病毒能够有效抑制SKOV3细胞中CXCR4的表达。结论：CXCR4-siRNA重组腺病毒载体可高效转染靶细胞和沉默CXCR4基因的表达，为RNA干扰技术应用于卵巢癌的临床基因治疗奠定基础。
[Abstract]:The first part was to construct the recombinant adenovirus vector of CXCR4-siRNA by using AdMax vector system. Objective: to construct the recombinant adenovirus vector of CXCR4-siRNA by using AdMax vector system, and to study the effect of the recombinant adenovirus vector on the expression of CXCR4 gene in human ovarian cancer cell line SKOV3. Methods: the small interfering fragment of CXCR4 gene was cloned into the adenovirus shuttle plasmid PAVsi1.1.The recombinant shuttle plasmid was screened by PCR and sequenced. The recombinant shuttle plasmid and adenovirus-assisted packaging plasmid were co-transfected into HEK293 cells to produce CXCR4-siRNA recombinant adenovirus vector. The virus was packed and amplified in HEK293 cells, the virus solution was collected and the titer of the virus was measured. Results the small interfering fragment of CXCR4 gene was successfully constructed into adenovirus shuttle plasmid. After repeated HEK293 packaging and amplification, a large amount of green fluorescent protein expression in HEK293 cells was observed under fluorescence microscope. The titer of the virus was 6 脳 108 PFU / ml. Conclusion the CXCR4-siRNA recombinant adenovirus vector can be constructed successfully by using the CXCR4-siRNA vector system. The second part of the study on the silencing of CXCR4 gene expression in human ovarian cancer cell line by CXCR4-siRNA recombinant adenovirus vector objective: to study the effect of CXCR4-siRNA recombinant adenovirus vector on the expression of CXCR4 gene in human ovarian cancer cell line SKOV3. Methods: a large number of viral fluids were transfected into human ovarian cancer cell line SKOV3. The cells were divided into three groups: the negative control group transfected with empty virus vector and the blank control group without any treatment. The silencing effect of CXCR4 gene was detected by immunohistochemistry. Results: after the virus was transfected into SKOV3 cells, a large amount of green fluorescent protein expression in SKOV3 cells was observed under fluorescence microscope, and the recombinant adenovirus could effectively inhibit the expression of CXCR4 in SKOV3 cells. Conclusion the recombinant adenovirus vector of CXCR4-siRNA can efficiently transfect target cells and silence the expression of CXCR4 gene, which lays a foundation for the application of RNA interference technique in clinical gene therapy of ovarian cancer.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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