| Osteopontin特异性siRNA慢病毒表达载体的构建及其在U251胶质瘤细胞中OPN的表达 |
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| 引用本文： | 晋雯,黄智勇,高美钦,李赞.Osteopontin特异性siRNA慢病毒表达载体的构建及其在U251胶质瘤细胞中OPN的表达[J].第四军医大学学报,2009(14). |
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| 作者姓名： | 晋雯 黄智勇 高美钦 李赞 |
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| 作者单位： | 福建医科大学基础医学院病理学系;福建医科大学基础医学院神经生物中心;福建医科大学基础医学院肿瘤研究室; |
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| 基金项目： | 福建医科大学神经生物学基金(0160003) |
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| 摘 要： | 目的:观察慢病毒表达载体介导的RNA干扰(RNAinterference,RNAi)对人胶质瘤细胞U251骨桥蛋白(osteopon-tin,OPN)表达的影响,为后续的以OPN基因为靶点的神经胶质瘤基因治疗研究奠定基础.方法:应用基因工程技术,筛选出2条针对OPN基因的RNAi靶序列,分别与pGCL-GFP载体连接,构建2个重组慢病毒表达载体LV-OPNshRNA1,LV-OPNshRNA2;将连接产物转化到DH5α感受态细胞,经PCR筛选阳性克隆、测序鉴定.将LV-OPNshRNA,pHelper 1.0,pHelper 2.0共转染293T细胞,包装产生慢病毒颗粒并测定病毒滴度.将包装产生的2种重组慢病毒分别感染U251细胞,实时定量PCR和Western印迹检测U251细胞OPN mRNA和蛋白的表达,并与未转染及空转染细胞进行比较.结果:2个慢病毒载体PCR和测序结果与预期结果一致,经包装产生的病毒滴度为8×1010TU/L和5×1010TU/L.感染U251细胞后,OPN基因mRNA和蛋白的表达量与未感染慢病毒的细胞组及空载体感染组相比均明显下降.结论:成功构建针对OPN基因的2个慢病毒载体OPN...
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| 关 键 词： | RNA干扰 骨桥蛋白 慢病毒属 神经胶质瘤 |
Construction of lentiviral vector of siRNA specific for osteopontin and its expression in U251 glioma cell line |
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| JIN Wen,HUANG Zhi-Yong,GAO Mei-Qing,LI Zan.Construction of lentiviral vector of siRNA specific for osteopontin and its expression in U251 glioma cell line[J].Journal of the Fourth Military Medical University,2009(14). |
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| Authors: | JIN Wen HUANG Zhi-Yong GAO Mei-Qing LI Zan |
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| Institution: | JIN Wen1,3,HUANG Zhi-Yong1,GAO Mei-Qing1,2,LI Zan1,2 1Department of Pathology,Fujian Medical University,2Institute of Oncology,3Institute of Neurobiology,Fuzhou 350001,China |
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| Abstract: | AIM: To observe the influence of lentiviral vector-mediated RNA interference on expression of human osteopontin(OPN) gene in human glioma cell line U251 so as to pave a way for OPN gene-targeted gene therapy of glioma.METHODS:Gene engineering technique was used to screen 2 RNA interference sequences targeting OPN gene.The sequences were separately cloned into the pGCL-GFP vector to construct LV-OPNshRNA1 and LV-OPNshRNA2,which were subsequently confirmed by PCR and DNA sequencing analysis.The titer of lenti... |
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| Keywords: | RNA interference osteopontin lentivirus glioma |
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