机械敏感离子通道Trpm7在LIPUS促进牙髓间充质干细胞成骨分化中的作用机制的研究  

Mechanically Sensitive Ion Channels Trpm7 in LIPUS Promotes Interdental Dentinal Mesenchyme Study on the Mechanism of Action in Osteogenic Differentiation

作　　者：姚欢 潘华锋 郑妍[1] 章杰 李娅莎[1] 吴梦云 杨珂[1] Yao Huan;Pan Huafeng;Zheng Yan;Zhang Jie;Li Yasha;Wu Mengyun;Yang Ke(Key Laboratory of Children＇s Developmental Diseases Research,Ministry of Education,Children＇s Hospital of Chongqing Medical Universit;National International Science and Technology Cooperation Base for Major Children＇s Development,＂ Chongqing Key Laboratory of Pediatric;Chongqing Stem Cell Therapy Engineering Research Center,Chongqing Cell Bioengineering Technology Co.,Ltd.,Chongqing 400014,China)

机构地区：[1]重庆医科大学附属儿童医院儿童发育疾病研究教育部重点实验室儿童发育重大疾病国家国际科技合作基地儿科学重庆市重点实验室重庆市干细胞治疗工程技术研究中心重庆市细胞生物工程技术有限公司,重庆400014

出　　处：《中国细胞生物学学报》2018年第9期1518-1525,共8页Chinese Journal of Cell Biology

基　　金：重庆市自然科学基金一般项目(批准号:cstc2017jcyj AX0171;cstc2014jcyj A10090)资助的课题~~

摘　　要：该文主要研究低强度脉冲超声(low-intensity pulsed ultrasound,LIPUS)促进牙髓间充质干细胞(dental pulp mesenchymal stem cells,DPSCs)成骨分化,以及瞬时受体电位M7(transient receptor potential melastatin 7,Trpm7)在其中发挥的作用。培养人DPSCs,流式细胞术检测其表面分子标志表达,阿利新蓝、茜素红及油红O染色检测其成软骨、成骨和成脂分化能力。ALP活性、ALP染色和茜素红染色观察LIPUS促成骨分化的能力。实时定量PCR检测LIPUS处理组与对照组成骨分化相关基因OPN、OCN、RUNX2表达的差异以及不同时间点两组Trpm7 m RNA表达水平的变化。ALP活性检测LIPUS及不同浓度Trpm7抑制剂2-氨基乙酯二苯基硼酸(2-aminoethoxydiphenyl borate,2-APB)对成骨分化能力的影响。实验分为对照组、LIPUS组、LIPUS+二甲基亚砜(DMSO)组和LIPUS+2-APB组,ALP和茜素红染色观察各组成骨分化能力,Western blot检测各组OPN、OCN和RUNX2的蛋白表达。结果显示,成功培养DPSCs,LIPUS处理后ALP和茜素红阳性染色明显增多,ALP活性增强(P<0.01);OPN、OCN、RUNX2的m RNA表达水平显著增加(P<0.05),LIPUS处理第2天和第5天Trpm7的m RNA表达水平有明显升高(P<0.05)。2-APB作用后明显下调ALP活性(P<0.01)。LIPUS组、LIPUS+DMSO组与对照组相比,ALP和茜素红阳性染色以及OPN、OCN与RUNX2的蛋白表达均显著增加,而LIPUS+2-APB组较于LIPUS+DMSO组,ALP和茜素红染色以及OPN、OCN与RUNX2的蛋白表达明显降低。该研究结果提示,LIPUS能够促进DPSCs的成骨分化,且Trpm7在这一过程中发挥着重要作用。The purpose of this study was to investigate the role of low-intensity pulsed ultrasound（LIPUS） in promoting osteogenic differentiation of dental pulp mesenchymal stem cells（DPSCs） and the role of transient receptor potential M7（Transient receptor potential melastatin 7, Trpm7） involved. The human DPSCs were cultured. The surface molecular markers were detected by flow cytometry. The alcian blue, alizarin red and oil red O staining were used to detect the chondrogenic, osteogenic and adipogenic differentiation ability. ALP activity, ALP staining and alizarin red staining were observed the ability of LIPUS to promote osteogenic differentiation. The Real-time quantitative PCR was used to detect the difference of OPN, OCN and RUNX2 expression in the LIPUS-treated group and the control group, and the m RNA expression levels of Trpm7 in the two groups were detected at different time points. ALP activity was used to examine the effect of LIPUS and different concentrations of Trpm7 inhibitor 2-Aminoethoxydiphenyl borate（2-APB） on osteogenic differentiation. The osteogenic differentiation ability was observed by ALP staining and alizarin red staining in control group, LIPUS group, LIPUS＋DMSO group and LIPUS＋2-APB group. The expressions of OPN, OCN and RUNX2 were detected by Western blot. The results showed the cultured cells were identified as DPSCs. In the LIPUS treatment group, ALP staining and alizarin red staining increased significantly, and the ALP activity enhanced（P〈0.01）. The m RNA expression of OPN, OCN and RUNX2 were significantly increased（P〈0.05）. The m RNA expression of Trpm7 was significantly increased on the 5 th day（P〈0.05）. 2-APB significantly inhibited ALP activity（P〈0.01）. Compared with the control group, ALP staining, alizarin red staining and the protein expression of OPN, OCN, RUNX2 were significantly increased in LIPUS group and LIPUS＋DMSO group. Compared to LIPUS＋DMSO group, ALP and alizarin red staining, the protein expression of OPN, OCN, RUNX2 we

关 键 词：低强度脉冲超声 人牙髓间充质干细胞 Trpm7 成骨分化 

分 类 号：R683[医药卫生—骨科学]