上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (6): 753-.doi: 10.3969/j.issn.1674-8115.2011.06.016

• 论著（基础研究） • 上一篇    下一篇

慢病毒介导BMP2和VEGF165基因共转染对骨髓基质干细胞成骨分化的影响

蒋 佳1, 范存义2, 曾炳芳2   

  1. 1.复旦大学运动医学中心 华山医院运动医学与关节镜外科, 上海 200040； 2.上海交通大学附属第六人民医院骨科, 上海 200233
  • 出版日期:2011-06-28 发布日期:2011-06-27
  • 通讯作者: 曾炳芳, 电子信箱: bingfangz@gmail.com。
  • 作者简介:蒋 佳 (1981—), 女, 主治医师, 博士；电子信箱: jessicajj19@hotmail.com。
  • 基金资助:

    上海市科委重大项目(054119520)

Effects of lentivirus-mediated co-transfection of BMP2 gene and VEGF165 gene on osteogenic differentiation of bone marrow-derived mesenchymal stromal cells

JIANG Jia1, FAN Cun-yi2, ZENG Bing-fang2   

  1. 1.Sports Medicine Center of Fudan University, Department of Sports Medicine and Arthroscopy, Huashan Hospital, Shanghai 200040, China；2.Department of Orthopedics, the Sixth People's Hospital, Shanghai Jiaotong University, Shanghai 200233, China
  • Online:2011-06-28 Published:2011-06-27
  • Supported by:

    Shanghai Science and Technology Committee Foundation, 054119520

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摘要：

目的 观察慢病毒介导骨形成蛋白2 (BMP2)和血管内皮生长因子165（VEGF165）基因共转染对骨髓基质干细胞(MSCs)成骨分化的影响。方法 构建携带BMP2、VEGF165和绿色荧光蛋白（GFP）基因的重组慢病毒表达载体，包装、生产携带BMP2、VEGF165和GFP基因的重组慢病毒（Lv-BMP、Lv-VEGF、Lv-GFP）。分离、培养Wistar大鼠骨髓MSCs，分别以Lv-GFP(GFP组）、Lv-BMP（BMP组）、Lv-VEGF（VEGF组）转染MSCs，Lv-BMP和Lv-VEGF共转染MSCs（BMP+VEGF组），另设未转染MSCs对照组。RT-PCR法检测转染后7 d各组细胞BMP2、VEGF165 mRNA表达以及转染后1、2、4周各组细胞骨钙素(OCN) mRNA表达；ELISA法检测转染后1、4、8周各组细胞培养上清液BMP2、VEGF165蛋白表达以及转染后1、2、4周各组细胞培养上清液OCN蛋白表达；转染后第14天，各组细胞行碱性磷酸酶（ALP）染色和ALP活性检测。结果 BMP+VEGF组BMP2、VEGF165 mRNA和蛋白高效共表达，BMP2 mRNA和蛋白表达与BMP组比较差异无统计学意义(P>0.05)，VEGF165 mRNA和蛋白表达与VEGF组比较差异无统计学意义(P>0.05)；对照组、GFP组和VEGF组无BMP2 mRNA和蛋白表达；对照组、GFP组和BMP组无VEGF165 mRNA和蛋白表达；BMP+VEGF组OCN mRNA和蛋白表达显著高于其他各组(P<0.01)。BMP+VEGF组ALP染色阳性区域大于对照组、VEGF组和BMP组；BMP+VEGF组ALP活性显著高于其他各组(P<0.01)。结论 慢病毒介导BMP2和VEGF165基因共转染可促进骨髓MSCs成骨分化。

关键词: 骨形成蛋白2, 血管内皮生长因子165, 共转染, 慢病毒载体, 成骨分化

Abstract:

Objective To investigate the effects of lentivirus-mediated co-transfection of bone morphogenetic protein 2 (BMP2) gene and vascular endothelial growth factor 165 (VEGF165) gene on osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (MSCs). Methods The expression lentivirus vectors carrying VEGF165，BMP2 or green fluorescent protein (GFP) gene were constructed respectively, and recombinant lentivirus carrying VEGF165 (Lv-VEGF), BMP2 (Lv-BMP) or GFP (Lv-GFP) were packaged and produced respectively. Rat bone marrow-derived MSCs were isolated and cultured in vitro, and were transfected with Lv-VEGF (VEGF group), Lv-BMP (BMP group) or Lv-GFP (GFP group), co-transfected with Lv-VEGF and Lv-BMP (BMP+VEGF group), or transfected with no virus (control group). The expression of VEGF165 and BMP2 mRNA 7 d after transfection and that of osteocalcin (OCN) mRNA 1, 2 and 4 weeks after transfection in MSCs was detected by RT-PCR in each group. The expression of VEGF165 and BMP2 protein 1, 4 and 8 weeks after transfection and that of OCN protein 1, 2 and 4 weeks after transfection in supernatant fluid of culture was detected by ELISA in each group. Alkaline phosphatase (ALP) staining was conducted, and ALP activity of MSCs was measured 14 d after transfection in each group. Results VEGF165 and BMP2 mRNA and protein effectively coexpressed in BMP+VEGF group. There was no significant difference in the expression of BMP2 mRNA and protein between BMP+VEGF group and BMP group (P>0.05), and there was also no significant difference in the expression of VEGF165 mRNA and protein between BMP+VEGF group and VEGF group (P>0.05). There was no expression of BMP2 mRNA and protein in control group, GFP group and VEGF group, and there was also no expression of VEGF165 mRNA and protein in control group, GFP group and BMP group. The expression of OCN mRNA and protein in BMP+VEGF group was significantly higher than that in the other groups (P<0.01). The area with positive staining of ALP in BMP+VEGF group was larger than that in control group, VEGF group and BMP group, and the ALP activity in BMP+VEGF group was significantly higher than that in the other groups (P<0.01). Conclusion Lentivirus-mediated co-transfection of BMP2 gene and VEGF165 gene may promote osteogenic differentiation of bone marrow-derived MSCs.

Key words: bone morphogenetic protein 2, vascular endothelial growth factor 165, co-transfection, lentiviral vector, osteogenic differentiation