糖皮质激素影响间充质干细胞成骨分化的实验研究

Experimental study of the effect of glucocorticoids on the osteogenic differentiation of mesenchymal stem cells

目的探讨Notch信号通路在糖皮质激素影响间充质干细胞(mesenchymal stem cells, MSCs)成骨分化过程中的作用。方法培养MSCs,给予糖皮质激素(glucocorticoids, GCs)处理,计算凋亡率,分析细胞凋亡情况;通过real-time PCR方法,检测细胞内Notch信号通路靶基因Hes1和成骨相关基因(ALP,Col1agen1)mRNA的表达;通过Western blot方法检测Notch信号通路靶基因Hes1和成骨相关基因蛋白的表达;应用茜素红染色,检测细胞内成骨钙化作用。结果 GCs处理后,凋亡细胞增加[(80±5.789)vs对照组(60±7.132),P<0.05];MSCs成骨分化相关基因ALP、Collagen1mRNA和蛋白表达明显减低,与对照组相比,P<0.05;茜素红染色显示GCs组染色强度与对照组相比显著降低(P<0.05)。同时,Notch信号通路靶基因Hes1表达明显降低(与对照组相比,P<0.05),Notch信号通路表达明显受到抑制。激活Notch信号通路,细胞凋亡百分率降低为72±2.169(与GCs组相比,P<0.05);成骨分化基因ALP、Collagen1mRNA和蛋白表达都较GCs组有所提高(P<0.05);茜素红染色显示GCs+JAG1组染色强度与GCs组相比明显提高(P<0.05)。结论 GCs通过抑制Notch信号通路的表达,进而抑制MSCs的成骨分化。

Objective To investigate the exact role of Notch signaling pathway in the inhibition of osteogenic differentiation of mesenchymal stem cells(MSCs)by glucocorticoids(GCs).Methods MSCs were cultured.GCs were added to the medium.Cell apoptosis was determined using multi-mode microplate reader by 530 nm.The mRNA expression of Hes1 and osteoblastic markers(ALP,Collagen 1)was determined by real-time PCR.The protein of Hes1 and osteoblastic markers(ALP,Collagen 1)was determined by Western blot.The mineralization was detected by Alizarin red staining.Results After GCs treatment,the number of apoptotic cells was increased((80±5.789)vs Control(60±7.132),P<0.05)The mRNA and protein of osteogenic markers ALP and Collagen 1 was decreased compared with the control(P<0.05).The Alizarin staining density was decreased compared with the control(P<0.05).The expression of Notch target gene Hes1 was decreased significantly compared with the control(P<0.05)and the expression of Notch signaling pathway was decreased by GCs significantly.After activation of Notch signaling,the number of apoptotic cells was decreased to 72±2.169(compared with the GCs group,P<0.05).The mRNA and protein of osteogenic markers ALP and Collagen 1 was increased compared with the GCs group(P<0.05).The Alizarin staining density was increased in GCs+JAG1 group compared with the GCs group(P<0.05).Conclusion GCs demolish osteogenic differentiation of MSCs via Notch signaling pathway inhibition.