调控人骨髓间充质干细胞成骨分化的miR-106a及其靶基因骨形态发生受体蛋白2

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1、www.CRTER.org顾夙. 调控人骨髓间充质干细胞成骨分化的miR-106a及其靶基因骨形态发生受体蛋白2调控人骨髓间充质干细胞成骨分化的miR-106a及其靶基因骨形态发生受体蛋白2顾 夙(南阳市中心医院骨科三病区，河南省南阳市 473003)引用本文：顾夙. 调控人骨髓间充质干细胞成骨分化的miR-106a及其靶基因骨形态发生受体蛋白2J.中国组织工程研究，2016，20(28):4109-4116.DOI: 10.3969/j.issn.2095-4344.2016.28.001 ORCID: 0000-0002-7175-2213(顾夙)文章快速阅读：miR-106a通过骨形态发

2、生受体蛋白2调控骨髓间充质干细胞向成骨细胞的分化顾夙，男，1976年生，河南省南阳市人，1999年新乡医学院毕业，硕士，主治医师，主要从事骨髓干细胞方面的研究。中图分类号:R394.2文献标识码:A文章编号:2095-4344(2016)28-04109-08稿件接受：2016-05-13miR-106a双荧光素酶报告实验证实miR-106a直接靶向骨形态发生受体蛋白2转染miR-106a类似物Western blot实验证实miR-106a负调控骨形态发生受体蛋白2的表达siRNA敲除骨形态发生受体蛋白2碱性磷酸酶活性受到抑制RUNX2和OCN表达水平降低骨形态发生受体蛋白2 文题释义：成骨

3、细胞：是骨形成的主要功能细胞，负责骨基质的合成、分泌和矿化。骨不断地进行着重建，骨重建过程包括破骨细胞贴附在旧骨区域，分泌酸性物质溶解矿物质，分泌蛋白酶消化骨基质，形成骨吸收陷窝；其后，成骨细胞移行至被吸收部位，分泌骨基质，骨基质矿化而形成新骨。人骨髓间充质干细胞：是一种成体干细胞，不但能向中胚层的各种细胞如骨、软骨等分化，还能向外胚层的神经细胞、内胚层的消化和内分泌细胞如肝细胞和胰岛样细胞分化。1999年，Pittenger等首先用单细胞培养的方法获取了人骨髓基质细胞集落，进一步将此来自一个克隆的细胞分化为成骨细胞、软骨细胞和脂肪细胞。摘要背景：正常生理条件下，人骨髓间充质干细胞的骨生成和脂

4、肪生成维持平衡，成骨分化是骨骼发展的重要过程，骨骼形成需要成骨细胞的分化和成熟。目的：探索miR-106及其靶基因骨形态发生受体蛋白2在成骨分化中的作用，为后续研究奠定基础。方法：使用成骨诱导培养基诱导骨髓间充质干细胞分化为成骨细胞，通过Western blot和碱性磷酸酶染色检测成骨分化标志物表达情况判断成骨分化进程。通过转染模拟剂过表达miR-106a，转染siRNA敲降骨形态发生受体蛋白2的表达，分别使用Real-time PCR和Western blot检测miR-106a和骨形态发生受体蛋白2表达情况。TargetScan软件预测和双荧光素酶报告实验验证miR-106a和骨形态发生受

5、体蛋白2的相互作用。结果与结论：在成骨分化过程中miR-106a表达下调，骨形态发生受体蛋白2表达上调。过表达miR-106a，结果发现碱性磷酸酶活性下降，成骨分化标志蛋白Runx2和OCN表达量下降，同时骨形态发生受体蛋白2表达下调。使用TargetScan预测骨形态发生受体蛋白2可能是miR-106a的靶基因，双荧光素酶报告实验验证了预测结果。敲降骨形态发生受体蛋白2的表达，成骨分化受到抑制。结果表明miR-106a通过靶向骨形态发生受体蛋白2从而调控骨髓间充质干细胞向成骨细胞分化。关键词：干细胞；骨髓干细胞；骨形态发生受体蛋白2；miR-106a；骨髓间充质干细胞；成骨分化；分子机制；荧

6、光素酶报告；转染；碱性磷酸酶主题词：微RNAs；细胞分化；组织工程3 P.O.Box 1200,Shenyang 110004 kf23385083miR-106a regulates the osteogenic differentiation of human bone marrow mesenchymal stem cells by targeting bone morphogenetic protein receptor 2Gu Su (Third Ward, Department of Orthopedics, Nanyang City Central Hospital, Nanya

7、ng 473003, Henan Province, China)AbstractBACKGROUND: Under normal physiological conditions, there is a homeostasis between the osteogenic and adipogenic differentiation of human bone marrow mesenchymal stem cells. Osteogenic differentiation is an important process in the formation of skeleton in whi

8、ch differentiated and mature osteoblasts are indispensable.OBJECTIVE: To explore the role of miR-106a and its target gene, bone morphogenetic protein receptor 2 (BMPR2) in the differentiation of human bone marrow mesenchymal stem cells into osteoblasts.METHODS: Human bone marrow mesenchymal stem cel

9、ls were induced to differentiate into osteoblasts by osteoblast-specific induction medium, and this process was detected by western blot and alkaline phosphatase staining. Overexpression of miR-106a was elicited by transfecting miR-106a mimics and the BMPR2 knockdown achieved by RNA interference. Th

10、e expression levels of miR-106a and BMPR2 were detected by real-time PCR and western blot, respectively. The interaction of miR-106a and BMPR2 was verified by TargetScan software and dual luciferase report experiment assay. RESULTS AND CONCLUSION: The expression of miR-106a was decreased whereas the

11、 expression of BMPR2 increased with the progress of osteogenesis differentiation. When miR-106a was overexpressed, alkaline phosphatase activity was declined and the expressions of runt-related transcription factor 2 and osteocalcin, markers of osteogenesis differentiation, both decreased. The expre