miR-424在人脂肪来源间充质干细胞成骨分化中的调控作用

发布时间：2018-04-04 12:06

本文选题：人脂肪来源间充质干细胞　切入点：miR-　出处：《上海交通大学学报(医学版)》2016年07期

【摘要】：目的探讨miR-424在人脂肪来源间充质干细胞(h ASC)成骨分化中的表达及miR-424对h ASC体外成骨分化的生物学作用。方法 q PCR检测miR-424在h ASC成骨分化不同阶段的表达水平。通过慢病毒颗粒在体外感染h ASC,使miR-424过表达或抑制其表达,进行Western blotting、细胞免疫荧光试验及碱性磷酸酶(ALP)和茜素红(ARS)染色试验,研究miR-424对骨形态发生蛋白2(BMP-2)及成骨诱导液诱导h ASC成骨分化过程的影响。预测miR-424的靶基因,并利用q PCR及Western blotting验证。结果 q PCR结果显示miR-424在h ASC的表达量随着成骨诱导时间延长逐渐下降(P0.05)。在BMP-2诱导下,过表达组的碱性磷酸酶(ALP)和骨桥蛋白(OPN)蛋白水平均下降,而抑制组的表达增加(P0.05)。细胞免疫荧光显示,过表达miR-424的细胞骨唾液酸蛋白(BSP)阳性率显著下降(P0.05)。ALP和ARS染色显示,过表达组ALP活性降低,钙盐沉积下降,而抑制组ALP活性升高,矿化能力增强。过表达miR-424能降低h ASC中Smad5的mRNA和蛋白水平,而抑制miR-424则使其水平升高(P0.05)。结论 miR-424在h ASC成骨分化中表达下调,其在hASC体外成骨分化过程中起抑制作用。
[Abstract]:Objective to investigate the expression of miR-424 in osteogenic differentiation of human adipose derived mesenchymal stem cells (hASCs) and the biological effect of miR-424 on in vitro osteogenic differentiation of h ASC.Methods Q PCR was used to detect the expression of miR-424 in different stages of h ASC osteogenesis.MiR-424 was overexpressed or inhibited by infection of lentivirus particles in vitro with Western blotting, cellular immunofluorescence assay and alkaline phosphatase (ALP) and alizarin red ARS staining.To study the effects of miR-424 on bone morphogenetic protein 2 (BMP-2) and osteogenic induction fluid induced h ASC osteogenic differentiation.The target genes of miR-424 were predicted and verified by Q PCR and Western blotting.Results Q PCR showed that the expression of miR-424 in h ASC decreased gradually with the prolongation of osteogenic time (P 0.05).The levels of alkaline phosphatase (ALP) and osteopontin (OPN) protein decreased in the overexpression group, but increased in the inhibition group.Cell immunofluorescence showed that the positive rate of bone sialic acid protein (miR-424) in overexpression group decreased significantly (P 0.05). ALP and ARS staining showed that ALP activity decreased, calcium salt deposition decreased in overexpression group, ALP activity increased and mineralization ability increased in inhibition group.Overexpression of miR-424 could decrease the level of mRNA and protein of Smad5 in h ASC, while inhibiting miR-424 increased the level of Smad5.Conclusion the expression of miR-424 is down-regulated during h ASC osteogenic differentiation, and it plays an inhibitory role in the process of hASC osteogenic differentiation in vitro.
【作者单位】：上海交通大学医学院附属第九人民医院眼科;
【基金】：国家自然科学基金(81470663)~~
【分类号】：R779.6

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