不同机械牵张强度对骨髓间充质干细胞成骨分化及lncRNA H19的影响

The effects of different mechanical stretches on osteogenic differentiation of bone marrow mesenchymal stem cells and lncRNA H19

目的探究不同机械牵张强度对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨分化以及lncRNA H19表达的影响。方法提取C57BL/6小鼠原代BMSCs,进行培养和鉴定。用Flexcell 5000细胞应力加载系统进行机械牵张,方案为频率0.5 Hz,连续牵张7 d,牵张强度为4%和6%,单次牵张时间分别为0、2、4、6、8 h。干预结束后采用RT-PCR验证lncRNA H19和成骨标志物OCN mRNA相对表达量,通过Western Blot检测Runx2蛋白相对表达量、ALP染色检测成骨分化能力。结果4%和6%的机械牵张干预均能促进BMSCs的成骨分化及lncRNA H19表达,其在4%牵张强度下牵张6 h内持续升高,8 h出现下降;在6%牵张强度下牵张4 h内持续升高,6 h出现下降,但都仍高于0 h。结论在固定牵张强度刺激下,一定时间范围内,牵张时间的增加能促进BMSCs成骨分化,长时间的牵张不利于成骨分化。本研究确定的促成骨分化及lncRNA H19表达的最佳牵张方案为6%牵张强度,4 h/d,连续牵张7 d,频率0.5 Hz。

Objective To investigate the effects of different mechanical stretch intensity on osteogenic differentiation and lncRNA H19 expression of Bone marrow mesenchymal stem cells(BMSCs).Methods The primary BMSCs of C57BL/6 mice were extracted,cultured and identified.Mechanical stretching was performed with the Flexcell 5000 cell stress loading system,the program was frequency of 0.5 Hz,continuous stretching for 7 days,stretching intensity of 4%and 6%,and single stretching time of 0,2,4,6 and 8 h.After the intervention,RT-PCR was used to verify the relative expression of lncRNA H19 and osteogenic marker OCN mRNA,western blot was used to detect the relative expression of Runx2 protein,and ALP staining was used to detect the osteogenic differentiation ability.Results Both 4%and 6%mechanical distraction interventions could promote the osteogenic differentiation of BMSCs and the expression of lncRNA H19.At 4%tensile intensity,it continued to increase within 6 h and decreased at 8 h;At 6%tensile intensity,it continued to increase within 4 h and decreased at 6 h,but still higher than 0 h.Conclusion Under the stimulation of fixed distraction intensity,within a certain time range,the increase of distraction time can promote the osteogenic differentiation of BMSCs,while prolonged distraction is not conducive to osteogenic differentiation.The optimal distraction regimen for promoting osteogenic differentiation and lncRNA H19 expression determined in this experiment was 6%distraction intensity,4 h/day,continuous distraction for 7 days,and a frequency of 0.5 Hz.