干扰foxM1基因表达可增强骨髓间充质干细胞的成骨分化能力  

Knockdown of foxM1 strengthens osteogenic differentiation ability of bone marrow mesenchymal stem cells

作　　者：陈钦桂 曾勉[1] 何婉媚[1] 张莉珊 郑海崇 Chen Qingui;Zeng Mian;He Wanmei;Zhang Lishan;Zheng Haichong(Medical Intensive Care Unit, First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, Guangdong Province, China)

机构地区：[1]中山大学附属第一医院内科重症监护病房,广东省广州市510080

出　　处：《中国组织工程研究》2019年第5期673-679,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81670066);项目负责人:曾勉;广东省省级科技计划项目(2016A020216009);项目负责人:曾勉;贝朗蛇牌学院重症科学研究基金资助项目(2017);项目负责人:曾勉~~

摘　　要：背景:foxM1基因被认为参与了干细胞分化命运的调控,但其对间充质干细胞成骨分化的影响尚未见报道,关于foxM1基因的研究也主要集中于肿瘤领域。目的:探索干扰foxM1基因表达水平对骨髓间充质干细胞成骨分化能力的影响。方法:全骨髓贴壁法培养SD大鼠(中山大学实验动物中心提供)骨髓间充质干细胞,构建含嘌呤霉素抗性基因的foxM1 shRNA重组慢病毒载体并转染大鼠骨髓间充质干细胞,经嘌呤霉素筛选建立foxM1稳定敲低细胞株,另设置空病毒载体组以及未转染组。使用骨髓间充质干细胞成骨诱导分化培养基诱导培养并行茜素红染色检测其成骨分化能力,使用定量RT-PCR检测成骨相关基因的表达水平,同时提取胞核蛋白,Western blot检测β-catenin蛋白表达水平。结果与结论:(1)与空病毒载体组以及未转染组比较,foxM1敲低的大鼠骨髓间充质干细胞经成骨诱导分化后形成的骨结节数量明显增多,成骨相关基因col1a1和runx2表达水平均显著升高,但胞核β-catenin蛋白水平无明显改变;(2)干扰foxM1基因表达可增强骨髓间充质干细胞的成骨能力。BACKGROUND:FoxM1is thought to be involved in the regulation of stem cell differentiation fate,but its effect on osteogenic differentiation of mesenchymal stem cells has not been reported.The research on foxM1gene is mainly focused on the tumor field.OBJECTIVE:To investigate the effect of shRNA-mediated knockdown of foxM1on the osteogenic differentiation ability of bone marrow mesenchymal stem cells.METHODS:Bone marrow mesenchymal stem cells from Sprague-Dawley rats were isolated and cultured by the whole bone marrow adherence method.Recombinant lentivirus carrying foxM1-specific shRNA and puromycin-resistance gene was constructed and transfected into the rat bone marrow mesenchymal stem cells,and foxM1-knockdown bone marrow mesenchymal stem cells were acquired after puromycin screening.Alizarin red staining was performed to investigate the osteogenic differentiation ability of bone marrow mesenchymal stem cells cultured in osteogenic differentiation medium.Expression levels of several osteogenesis-related genes were examined using quantitative PCR.The nuclear expression ofβ-catenin was detected using western blot.RESULTS AND CONCLUSION:Knockdown of foxM1in bone marrow mesenchymal stem cells enhanced the capability to form mineralized nodules and significantly increased the mRNA expressions of col1a1and runx2,while no significant difference was found in the nuclear protein expressions ofβ-catenin.These results suggest that knockdown of foxM1can promote the osteogenic differentiation of bone marrow mesenchymal stem cells.

关 键 词：骨髓间充质干细胞 成骨 诱导分化 慢病毒载体 foxM1基因 基因干扰 β-catenin 国家自然科学基金 叉头转录因子类 RNA干扰 骨髓 间质干细胞 细胞分化 成骨细胞 组织工程 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]