骨质疏松症大鼠脂肪干细胞成骨能力及Mettl14和Notch1表达的探究

Osteogenic Capacity and Mettl14 and Notch1 Expression of Adipose-Derived Stem Cells from Osteoporotic Rats

目的比较骨质疏松症脂肪干细胞(osteoporosis adipose-derived stem cells,OP-ASCs)和正常脂肪干细胞(control adipose-derived stem cells,Ctrl-ASCs)成骨能力的差异,并探究RNA甲基化转移酶14(methyltransferase-like 14,Mettl14)以及Notch信号分子1(Notch signaling molecule 1,Notch1)的表达水平。方法去势法(OVX)构建骨质疏松症SD大鼠动物模型(OP组),同时设立对照组(Ctrl组)。Micro-CT、HE和Masson染色鉴定骨质疏松症模型是否构建成功。贴壁法分离培养出OP-ASCs和Ctrl-ASCs。茜素红、阿利辛蓝和油红O染色检测OP-ASCs和Ctrl-ASCs三向分化能力,流式细胞术检测OP-ASCs和Ctrl-ASCs表面抗原CD29、CD44、CD90、CD31、CD34、CD45鉴定脂肪干细胞(adipose-derived stem cells,ASCs)。茜素红染色及Runt相关转录因子2(Runt-related transcription factor 2,Runx2)mRNA和蛋白的表达比较OPASCs和Ctrl-ASCs成骨能力的差异。实时荧光定量PCR、Western blot探究OP-ASCs和Ctrl-ASCs的Mettl14、Notch1在mRNA和蛋白水平上的表达差异。结果Micro-CT、HE和Masson染色显示OP组胫骨较Ctrl组骨小梁数量减少,间距增大,证明OP组建模成功。三向分化及流式细胞检测结果证明成功分离培养ASCs。成骨诱导后,茜素红染色显示OP-ASCs矿化结节较Ctrl-ASCs数量少、分布散在;OP-ASCs中Runx2表达较Ctrl-ASCs低(P<0.05)。OP-ASCs的Mettl14以及Notch1表达较Ctrl-ASCs低(P<0.05)。结论OP-ASCs成骨能力较Ctrl-ASCs低,OP-ASCs的Mettl14表达降低,Notch信号通路受到抑制。为进一步研究OP-ASCs成骨分化过程中Mettl14和Notch1的具体作用机制奠定了基础。

Objective To investigate the differences in the osteogenic capacity of osteoporotic adipose-derived stem cells(OP-ASCs)and normal control adipose-derived stem cells(Ctrl-ASCs),and to examine the expression levels of RNA methyltransferase like 14(Mettl14)and the Notch signaling molecule 1(Notch1).Methods The osteoporosis(OP)model of SD rats was established with ovariectomy(OVX).Micro-CT,HE staining and Masson staining were performed to identify the successful establishment of the OP model,OP-ASCs and Ctrl-ASCs were isolated and cultured adherently.Then,the three-way differentiation capacity of the adipose-derived stem cells(ASCs)was determined through alizarin red staining,alcian blue staining and oil red O staining and flow cytometry was conducted to examine the surface antigens CD29,CD44,CD90,CD31,CD34,and CD45.Alizarin red staining and comparison of the mRNA and protein expression of Run-related transcription factor 2(Runx2)were done to explore the differences in osteogenic potential of OP-ASCs and Ctrl-ASCs.Real-time PCR and Western blot were performed to explore the expression differences of Mettl14 and Notch1 at mRNA and protein levels between OP-ASCs and Ctrl-ASCs.Results Micro-CT,HE and Masson staining results showed that the number of trabecular bone decreased and the spacing increased in the tibias of the osteoporosis group(OP group)compared with those of the control group(Ctrl group),indicating that the OP model was established successfully.Three-way differentiation and flow cytometry results confirmed the successful isolation and culture of ASCs.After osteogenic induction,alizarin red staining showed that OP-ASCs had fewer number and more scattered distribution of mineralized nodules than Ctrl-ASCs did.The expression of Runx2 in OP-ASCs was lower than that in CtrlASCs(P<0.05).Mettl14 as well as Notch1 showed lower expression in OP-ASCs than they did in Ctrl-ASCs(P<0.05).Conclusion The osteogenic capacity of OP-ASCs was lower compared with that of Ctrl-ASCs,Mettl14 expression of OP-ASCs was decreased compared with that of Ctrl-ASCs,and the Notch signaling pathway was inhibited in OP-ASCs.The study helps build the foundation for further investigation in the specific mechanisms of Mettl14 and Notch1 during osteogenic differentiation of OP-ASCs.