中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (40): 7541-7544.doi: 10.3969/j.issn.1673-8225.2011.40.030

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

Nesprin基因RNAi慢病毒载体的构建

杨文钢，薛  松，连  锋，汪永义，黄日太，朱洪生   

  1. 上海交通大学医学院附属仁济医院心胸外科，上海市  200127
  • 收稿日期:2011-07-05 修回日期:2011-08-06 出版日期:2011-10-01 发布日期:2011-10-01
  • 通讯作者: 薛松，主任医师，博士生导师，上海交通大学医学院附属仁济医院心胸外科，上海市 200127 XUESONG64@163.COM
  • 作者简介:杨文钢★，男，1979年生，江西省玉山市县人，汉族，2010年上海交通大学医学院毕业，硕士，医师，主要从事冠心病的外科治疗与干细胞的基础研究。 ywgada@hotmail.com
  • 基金资助:

    上海市科委基金资助项目(064119636)。

Construction of Nesprin genome RNAi lentiviral vector

Yang Wen-gang, Xue Song, Lian Feng, Wang Yong-yi, Huang Ri-tai, Zhu Hong-sheng   

  1. Department of Cardiothoracic Surgery, Renji Hospital Affiliated to Medical College of Shanghai Jiao Tong University, Shanghai  200127, Jiangsu Province, China
  • Received:2011-07-05 Revised:2011-08-06 Online:2011-10-01 Published:2011-10-01
  • Contact: Xue Song, Chief physician, Doctoral supervisor, Department of Cardiothoracic Surgery, Renji Hospital Affiliated to Medical College of Shanghai Jiao Tong University, Shanghai 200127, Jiangsu Province, China XUESONG64@163.com
  • About author:Yang Wen-gang★, Master, Physician, Department of Cardiothoracic Surgery, Renji Hospital Affiliated to Medical College of Shanghai Jiao Tong University, Shanghai 200127, Jiangsu Province, China ywgada@hotmail.com
  • Supported by:

    Shanghai Science and Technology Committee, No. 064119636*

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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

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 DNA identified by sequence. siRNA interference with the four kinds of plasmids (SR-1, SR-2, SR-3, SR-4) were transformed into rat vascular smooth muscle cells to screen the most effective sequence; In order to get the sequence started with interfering carrier by RT-PCR and western-blot, we let the best interfering sequence carriers and pDONR221 to react together. Then entry vectors and lentiviral vectors to express the purpose of pLenti6/V5-DEST response was to restructure the sequence containing interference lentiviral expression vector. Lentiviral vector containing interfering sequence was co-transfected 293T cells to package lentivirus. The virus titer was determined by the expression level of GFP protein in 293T cells. Lentivirus was used to transfect rat bone marrow mesenchymal stem cells.
RESULTS AND CONCLUSION: Sequencing confirmed the successful construction of Nesprin siRNA lentiviral vector LV-siNesprin. The best interference with miRNA plasmid selected by RT-PCR and western-blot was SR-3. The virus titer for lentiviral packaging and concentrating suspension of the activity was 106 TU / ml. The expression of Nesprin protein decreased in transfected cells.By certain conditions, Nesprin-siRNA lentiviral vector can be successfully constructed.

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