Hey1表达对BMP-9诱导下C3H10T1/2细胞的成骨分化及增殖影响

EFFECT OF Hey1 EXPRESSION ON OSTEOGENIC DIFFERENTIATION AND PROLIFERATION OF C3H10T1/2 CELLS INDUCED BY BONE MORPHOGENETIC PROTEIN 9

目的探讨Notch信号通路重要靶点Hey1表达水平改变对BMP-9诱导C3H10T1/2细胞成骨分化与增殖的影响。方法构建过表达Hey1慢病毒LV-Hey1、抑制Hey1表达慢病毒LV-sh Hey1,分别感染C3H10T1/2细胞干预Hey1表达水平,以LV-Blank(空质粒)感染C3H10T1/2细胞作为对照;以荧光显微镜对慢病毒感染效果、实时荧光定量PCR以及Western blot对Hey1表达水平进行验证,筛选不同Hey1表达水平的稳定细胞系。用含BMP-9的条件培养基诱导不同Hey1表达水平的C3H10T1/2细胞(分别为BMP-9+C3H10T1/2组、BMP-9+C3H10T1/2-Hey1组、BMP-9+C3H10T1/2-sh Hey1组),以正常培养基培养的细胞作为对照(C3H10T1/2组、C3H10T1/2-Blank组)。培养后48 h,实时荧光定量PCR及Western blot测定成骨分化相关转录因子Runx2、骨桥蛋白、骨钙素m RNA及蛋白表达水平;4、5、6、7 d行MTT检测及4、5、10 d行流式细胞仪测定细胞增殖能力;4、7 d时ELISA测定细胞ALP表达水平并行染色观察。结果成功建立不同Hey1表达水平稳定细胞系。成骨方面,各时间点与BMP-9+C3H10T1/2组比较,BMP-9诱导下Hey1过表达的BMP-9+C3H10T1/2-Hey1组细胞Runx2、骨桥蛋白、骨钙素m RNA及蛋白表达水平以及成骨分化标志物ALP含量均显著增加(P<0.05),抑制Hey1表达的BMP-9+C3H10T1/2-sh Hey1组细胞以上指标均显著降低(P<0.05)。对细胞增殖活力影响方面,与BMP-9+C3H10T1/2组比较,BMP-9+C3H10T1/2-Hey1组MTT检测吸光度(A)值及细胞G2+S期百分比均提高(P<0.05);而抑制Hey1表达BMP-9+C3H10T1/2-sh Hey1组以上指标均降低(P<0.05)。结论 Hey1表达是BMP-9诱导C3H10T1/2细胞成骨分化重要环节,同时影响细胞早期增殖。

Objective To investigate the effect of Notch signaling pathway important target Hey1 expression on the differentiation and proliferation of C3H10T1/2 cells induced by bone morphogenetic protein 9（BMP-9）. Methods Hey1 lentivirus and Hey1 short hairpin RNA lentivirus were constructed and used to infect C3H10T1/2 cells to change the expression level of Hey1 in C3H10T1/2 cells. C3H10T1/2 cells infected with LV-Blank（empty plasmid） as control. The Hey1 expression levels of different groups were detected by fluorescence microscope, realtime fluorescence quantitative PCR, and Western blot. The C3H10T1/2 cells with different Hey1 expression level were induced by BMP-9 conditioned medium（BMP-9＋C3H10T1/2 group, BMP-9＋C3H10T1/2-Hey1 group, and BMP-9＋C3H10T1/2-shHey1 group）; the cells of control groups（C3H10T1/2 group and C3H10T1/2-Blank group） were cultured with normal medium. The mRNA and protein expression levels of osteogenesis related transcription factors（Runx2, osteopontin, and osteocalcin） were detected at 48 hours by real-time fluorescence quantitative PCR and Western blot assay. The cells proliferation and cycles were detected by MTT assay at 4, 5, 6, and 7 days and flow cytometry at 4, 5, and 10 days. The alkaline phosphatase（ALP） activity was analyzed by ELISA and observed by ALP staining at 4 and 7 days. Results C3H10T1/2 cell lines with different Hey1 expression levels were successfully established. In osteogenesis compared with BMP-9＋C3H10T1/2 group, overexpression of Hey1 enhanced the mRNA and protein expressions of transcription factors（Runx2, osteopontin, and osteocalcin）, and the expression of osteogenic differentiation marker（ALP）（P〈0.05）; however, inhibition of Hey1 expression significantly decreased the above indexes（P〈0.05）. In cell proliferation activity compared with BMP-9＋C3H10T1/2 group, overexpression of Hey1 increased absorbance（A） value in MTT assay and pecentage of G2＋S cells in cytometry assay, but inhibition of Hey1 expression significantly decreased the indexes（P〈0.05）. Conclusion Expression of Hey1 is the important link in the osteogenic differentiation process of C3H10T1/2 cells induced by BMP-9, and plays an important role in the regulation of early cell proliferation.