康 健,侯 洋,周许辉,杨立利,陈华江,唐先业,袁 文.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[J].中国脊柱脊髓杂志,2014,(2):157-163. |
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 |
中文关键词: TGF-β3 BMP-7 骨髓间充质干细胞 腺病毒 种子细胞 |
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。 |
Differentiation of rabbit bone marrow mesenchyme stem cells co-transfected by TGF-β3 and BMP-7 adenoviral vectors into nucleus pulposus-like cells |
英文关键词:BMSCs Adenovirus TGF-β3 BMP-7 Seed cell |
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. |
投稿时间:2013-04-09 修订日期:2013-12-16 |
DOI: |
基金项目:国家自然科学基金资助项目(81071509) |
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