TGF-β3和BMP-7腺病毒共转染兔骨髓间充质干细胞向类髓核细胞分化的研究

Differentiation of rabbit bone marrow mesenchyme stem cells co-transfected by TGF-β3 and BMP-7 adenoviral vectors into nucleus pulposus-like cells

中文关键词:  TGF-β3  BMP-7  骨髓间充质干细胞  腺病毒  种子细胞

英文关键词:BMSCs  Adenovirus  TGF-β3  BMP-7  Seed cell

中文摘要:

  【摘要】 目的：探讨利用转基因技术构建的转化生长因子β3（transforming growth factor beta 3，TGF-β3）和骨形态发生蛋白7（bone morphogenetic protein 7，BMP-7）重组腺病毒转染兔骨髓间充质干细胞（bone marrow mesenchyme stem cells，BMSCs），诱导其向类髓核细胞分化的可行性。方法：将培养获得的BMSCs分成5组，A：空白对照组，B：免疫荧光对照组，C：TGF-β3单独转染组，D：BMP-7单独转染组，E：TGF-β3+BMP-7共转染组。利用转基因技术构建的TGF-β3和BMP-7重组腺病毒对BMSCs进行转染，荧光显微镜观察其转染效率，培养14d后，再以Western-Blot方法检测A、C、D、E组细胞TGF-β3和BMP-7蛋白分泌情况，以Realtime PCR方法检测各组蛋白聚糖（ACAN）、Ⅰ型胶原（Collagen Ⅰ）、Ⅱ型胶原（Collagen Ⅱ）、Ⅹ型胶原（Collagen Ⅹ）、SOX9基因表达水平，明确腺病毒转染后兔骨髓间充质干细胞的分化情况。结果：以TGF-β3和BMP-7重组腺病毒转染兔BMSCs后常规DMEM培养基培养14d，细胞形态出现明显变化，圆形及椭圆形细胞明显增多。Western blot方法检测，共转染组TGF-β3和BMP-7蛋白表达水平明显增高。培养14d时，Realtime PCR检测ACAN、Collagen Ⅰ、Collagen Ⅱ、SOX9基因的表达水平转染组（C、D、E组）较对照组（A、B组）明显升高（P<0.05），其中Collagen Ⅰ和SOX9表达单独转染组（C、D组）与共转染组（E组）比较差异无显著性（P>0.05），Collagen Ⅱ表达共转染组较单独转染组明显增高（P<0.05）。TGF-β3单独转染组和共转染组的Collagen Ⅹ基因表达较BMP-7单独转染组和对照组明显降低（P<0.05）。结论：转基因技术重组TGF-β3和BMP-7腺病毒可成功转染兔BMSCs，获得TGF-β3和BMP-7蛋白的表达。TGF-β3和BMP-7腺病毒共转染兔BMSCs后，可有效诱导兔BMSCs向类髓核细胞方向分化。

英文摘要:

  【Abstract】 Objectives: To induce rabbit bone marrow mesenchyme stem cells(BMSCs) differentiating into nucleus pulposus-like cells(NPCs) by co-transfecting with TGF-β3 and BMP-7 adenoviral vectors. Methods: BMSCs were divided into five groups: A, blank control group; B, transfected by blank GFP adenoviral vector group; C, transfected by TGF-β3 adenoviral vector group; D, transfected by BMP-7 adenoviral vector group; E, co-transfected by TGF-β3 and BMP-7 adenoviral vectors group. The transfection result was observed by fluorescence microscope. After 14 days′ cultivation, the expressions of TGF-β3 and BMP-7 protein(group A, C, D, E) and ACAN, Collagen Ⅰ, Collagen Ⅱ, Collagen Ⅹ and SOX9(all group) in mRNA level were measured by Western blot and Realtime PCR respcetively. Results: 14 days after culture of co-transfected BMSCs, the shape of most cells changed obviously. Western blot showed higher expression of TGF-β3 and BMP-7 proteins in group E. The expressions of ACAN, Collagen Ⅰ, Collagen Ⅱ and SOX9 gene were much higher than those control group(group A, B)(P<0.05), and the expression of Collagen Ⅱ in co-transfected group was higher compared with single-transfected groups(P<0.05). The expression of Collagen Ⅹ in TGF-β3 transfected group and co-transfected group was obviously lower than that of BMP-7 transfected group and control group(P<0.05). Conclusions: The rabbit BMSCs are able to express TGF-β3 and BMP-7 proteins and differentiate into NPCs after co-transfected by TGF-β3 and BMP-7 adenoviral vectors.