牵张应力刺激介导间充质干细胞的信使RNA和长链非编码RNA表达谱变化

目的探讨信使RNA和长链非编码RNA在牵张应力刺激间充质干细胞（MSC）成骨分化中的作用。 方法分离培养正常人骨髓MSC，分为应力组与无应力组，应力组通过FlexCell应力系统对其施加外源性应力（5﹪形变、频率0.1 Hz、作用时间4 h/d），无应力组不予以外源性应力刺激，通过茜素红实验和碱性磷酸酶实验检测其成骨分化能力改变。提取应力刺激下第7天RNA，进行全基因组及长链非编码芯片表达谱检测，通过生物信息学方法分析和鉴定关键长链非编码RNA。采用两样本t检验进行统计学分析。 结果成骨第14天应力组MSC茜素红定量OD值为1.46±0.19，高于无应力组MSC 0.62±0.08，差异具有统计学意义（t?= -1.99，P < 0.05）。此外，成骨第14天应力组MSC碱性磷酸酶结果（265.3±31.2）U/L，较无应力组（121.2±21.2）U/L升高（t = -12.23，P < 0.05）。通过芯片表达谱检测，牵张应力刺激下成骨第7天共有差异表达信使RNA 598个，差异表达长链非编码RNA 329个。通过KEGG分析提示WNT信号通路是差异表达最明显的信号通路，其中WNT5a表达变化最为明显（6.74倍，P?< 0.01）。通过共表达分析提示lnc-RNA-SSR是调控WNT5a表达的主要长链非编码RNA。 结论牵张应力刺激下MSC的信使RNA和长链非编码RNA表达谱明显改变，其中lnc-RNA-SSR可能通过WNT5a影响MSC成骨分化能力。

Differentially expressed profile of messager RNA and long non-coding RNA in mesenchymal stem cell with strain stimulation

ObjectiveTo determine the role of mRNA and lnc-RNA in the osteogenic differentiation of MSC with strain stimulation. MethodsMSC from healthy donors were isolated, cultured and then divided into the experimental group and the control group. The experimental group was stimulated with strain using FlexCell system (5﹪, 0.1?Hz, 4?h/d), and the control group was cultured without stimulaiton. The osteogenic differentiation ability of MSC was detected by alizarin red and alkaline phosphatase assays. RNA was extracted on day 7 of osteogenic differentiation, which was then detected using the mRNA and lnc-RNA expression microarrays. The key lnc-RNAs were identified using bioinformatics technology. ResultsOn day 14 of osteogenic differentiation, the ARS quantitation results of MSC in the strain stimulation group (OD value: 1.46±0.19) were higher than those of the control group (OD value: 0.62±0.08). The difference was statistically significant (t?= -1.99, P < 0.05). Besides, the ALP activity results of MSC in the strain stimulation group (265.3±31.2)?U/L were also higher than MSC without strain stimulation (121.2±21.2)?U/L (t?= -12.23, P < 0.05) . Through microarray dection, a total of differentially expressed 598 mRNAs and 329 lnc-RNAs were identified on day 7 of osteogenic differentiation. KEGG analysis showed that WNT signal pathway was the key pathway with the most significant differences, among which WNT5a had the largest change. Co-expression analysis suggested that lnc-RNA-SSR may be the critical lnc-RNA to regulate WNT5a expression. ConclusionExpression profile of mRNA and lnc-RNA in MSC significantly changed with strain stimulation. Lnc-RNA-SSR may affected MSC osteogenesis through regulating WNT5a expression with strain stimulation