可控式转基因骨髓间充质干细胞复合β-磷酸三钙促成骨效应的实验研究

Experimental study on the osteogenic effect of adjustable transgenic BMSCs combined withβ-TCP

目的:观察与支架材料三维培养后,可控式碱性成纤维细胞生长因子(bFGF)联合音猬因子(Shh)的时序基因传递对骨髓间充质干细胞促成骨效应的调控作用。方法:使用受四环素调控的重组腺相关病毒rAAV2-tet-off-bFGF以及rAAV2-Shh共同感染大鼠骨髓间充质干细胞(BMSCs),体外与β-磷酸三钙(β-TCP)复合三维培养。扫描电镜观察细胞附着于材料表面的形貌。MTT检测复合物中细胞的增殖状况。实时定量PCR检测复合物中细胞内各成骨相关因子mRNA的表达变化。结果:扫描电镜可见三维培养后15 h两组细胞已黏附于材料表面,至培养后4 d,细胞呈多角形向材料内部孔隙伸展。MTT显示转基因BMSCs与β-TCP复合三维培养5 d后细胞增殖明显,与其它两组比较均有统计学差异。实时定量PCR及碱性磷酸酶检测发现双因子时序表达组,不管贴壁培养还是三维培养,胞内成骨相关因子的表达与对照组相比均有显著升高。结论:四环素调控下能成功实现bFGF及Shh在BMSCs中的时序表达,与β-TCP复合三维培养后,转基因细胞的增殖速度、成骨相关因子的表达均有显著提升。

Objective:To observe the osteogenic effect of bone marrow mesenchymal stem cells which combining with scaffold material after sequential delivered by bFGF and Shh.Methods:Rat bone marrow mesenchymal stem cells(BMSCs)were infected with rAAV2-Shh and rAAV2-tet-off-bFGF,the latter could be regulated by tetracycline,and then were combined withβ-tricalcium phosphate(β-TCP).The morphology of cells attached to the surface of the material was observed by scanning electron microscope(SEM).The proliferation of the cells in the complex was detected by MTT.The mRNA expression of various osteogenic factors in the complex were detected with real-time quantitative PCR.Results:SEM showed that two groups of cells had adhered to the surface ofβ-TCP after 15 hours of three-dimensional culture.After 4 days of culture,the cells showed a polygonal shape and extended to the inner pores of the material.After 5 days of three-dimensional culture,the cell proliferation was obviously increased in the group of transgenic BMSCs combiningβ-TCP,which was statistically different from the other two groups.Regardless of adherent culture or three-dimensional culture,the mRNA expression of intracellular osteogenic factors of bFGF/Shh sequential expression group were significantly higher than that of the control group.Conclusion:Under the control of tetracycline,the sequential expression of bFGF and Shh in BMSCs can be successfully achieved.After three-dimensional culturing withβ-TCP,the proliferation speed of transgenic cells and the expression of osteogenic factors are significantly improved.