| Nesprin基因RNAi慢病毒载体的构建 |
| |
| 引用本文： | 杨文钢,薛松,连锋,汪永义,黄日太,朱洪生.Nesprin基因RNAi慢病毒载体的构建[J].中国组织工程研究与临床康复,2011,15(40). |
| |
| 作者姓名： | 杨文钢 薛松 连锋 汪永义 黄日太 朱洪生 |
| |
| 作者单位： | 上海交通大学医学院附属仁济医院心胸外科,上海市,200127 |
| |
| 基金项目： | 上海市科委基金资助项目 |
| |
| 摘 要： | 背景:Nesprin蛋白缺失将影响细胞骨架组织和动态平衡,引起细胞骨架刚性丧失或导致细胞过早成熟老化,其对间充质干细胞的作用如何?目的:构建Nesprin蛋白siRNA慢病毒载体,并转染骨髓间充质干细胞.方法:针对Nesprin靶基因序列设计并合成4对miRNA oligo,将4种miRNA干扰质粒转入大鼠血管平滑肌细胞,筛选最有效干扰序列;将最佳干扰序列和pDONR221载体进行重组反应,获得含干扰序列的入门载体,再将入门载体和慢病毒表达目的载体pLenti6/V5-DEST进行重组反应,获得含干扰序列的慢病毒表达载体,转染包装细胞293T细胞,包装慢病毒,以293T细胞GFP蛋白水平测定病毒滴度.慢病毒转染大鼠骨髓间充质干细胞.结果与结论:测序证实合成的4对miRNA oligo正确,RT-PCR和western-blot筛选出最佳干扰miRNA质粒为SR-3,成功构建了Nesprin siRNA的慢病毒载体LV-siNesprin.包装慢病毒,浓缩病毒悬液的活性滴度为106 TU/mL.慢病毒成功了转染骨髓间充质干细胞细胞.
|
| 关 键 词： | 慢病毒载体 Nesprin蛋白 骨髓间充质干细胞 病毒滴度 干扰载体 |
Construction of Nesprin genome RNAi lentiviral vector |
| |
| Yang Wen-gang,Xue Song,Lian Feng,Wang Yong-yi,Huang Ri-tai,Zhu Hong-sheng.Construction of Nesprin genome RNAi lentiviral vector[J].Journal of Clinical Rehabilitative Tissue Engineering Research,2011,15(40). |
| |
| Authors: | Yang Wen-gang Xue Song Lian Feng Wang Yong-yi Huang Ri-tai Zhu Hong-sheng |
| |
| Institution: | Yang Wen-gang,Xue Song,Lian Feng,Wang Yong-yi,Huang Ri-tai,Zhu Hong-sheng Department of Cardiothoracic Surgery,Renji Hospital Affiliated to Medical College of Shanghai Jiao Tong University,Shanghai 200127,Jiangsu Province,China |
| |
| Abstract: | BACKGROUND: Nesprin protein absence can affect the cytoskeletal tissue and homeostasis, causing the loss of cytoskeleton rigidity or leading to premature aging of cells. In this study, we investigated the effect of Nesprin protein on bone marrow mesenchymal stem cells through designing Nesprin siRNA lentiviral vector. OBJECTIVE: To construct Nesprin-siRNA lentiviral vector. METHODS: According to the target gene sequence of Nesprin, four pairs of miRNA oligo were designed and annealed into double-stranded DN... |
| |
| Keywords: | |
| 本文献已被 CNKI 万方数据 等数据库收录！ |
|
