TERT基因修饰的绵羊脂肪间充质干细胞多向诱导分化的研究

Multi-direction Induced Differentiation Research of TERT Gene Modified Sheep Adipose Derived Mesenchymal Stem Cells

目的探讨TERT基因修饰的绵羊脂肪间充质干细胞在体外多向分化的能力,验证TERT基因对绵羊脂肪间充质干细胞永生化的作用。方法将TERT-ADSCs进行传代培养,选取P3和P30进行成骨和成神经的诱导分化,通过组织学染色和特异性表达基因的RT-PCR鉴定,对TERT-ADSCs的多向分化能力进行鉴定。结果 TERT-ADSCs可以稳定传至P30,P3和P30成骨诱导后,可见明显的骨结节突起,茜素红染色后呈红褐色,且表达特异性基因CBFA和Osteocalcin。成神经诱导后细胞呈三角或树突状形态,甲苯胺蓝染色后可见尼氏体存在,且表达特异性基因NSE和GFAP。结论绵羊TERT-ADSCs传至多代后仍然具有多向分化的潜能,为ADSCs永生化研究提供了理论基础。

Objective To investigate the in vitro multi-direction differentiation ability of TERT gene modified sheep adi-pose derived mesenchymal stem cells and to verify the role of TERT gene in immortalization of sheep adipose derived mesenchymal stem cells. Methods The TERT-ADSCs were subcultured. P3 and P30 cells were selected to proceed induced osteogenic and neu-ral differentiation. Histological staining and RT-PCR of specifically expressed genes were used to identify the multi-direction differ-entiation ability of TERT-ADSCs. Results TERT-ADSCs can be stably subcultured to P30. After induction of osteogenic differenti-ation of P3 and P30,process of bone nodules was obviously visible,cells were stained reddish brown after alizarin red staining and specific gene CBFA and Osteocalcin were expressed. After induction of neural differentiation,cells presented triangle or dendritic morphology,Nissl bodies can be observed by toluidine blue staining and specific gene NSE and GFAP were expressed. Conclusion The TERT-ADSCs of sheep still has the potential of multi-direction differentiation after having been subcultured several times, which provides a theoretical basis for the immortalization study of ADSCs.