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潘欣,曾思良,梁兴伦,等.MicroRNA-195-5p调节Bmpr1α表达对骨髓间充质干细胞成脂分化的影响[J].同济大学学报（医学版）,2017,38(3):1-7, 13. [点击复制]
- PAN Xin,ZENG Si-liang,LIANG Xing-lun,et al.MicroRNA-195-5p affects adipocyte differentiation in bone mesenchymal stem cells by regulating Bmpr1α gene expression[J].同济大学学报（医学版）,2017,38(3):1-7, 13. [点击复制]
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| MicroRNA-195-5p调节Bmpr1α表达对骨髓间充质干细胞成脂分化的影响 |
| 潘欣,曾思良,梁兴伦,嵇承栋,刘晓东,廖万清 |
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| (同济大学附属杨浦医院中心实验室,上海 200090;上海俊维寓医医院有限公司检验科,上海 200433;上海师范大学天华学院康复治疗学系,上海 201815;第二军医大学附属长征医院皮肤科,上海 200003) |
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| 摘要: |
| 目的 探讨miR-195-5p在补肾方含药血清干预大鼠原代骨髓间充质干细胞(bone mesenchymal stem cell, BMSC)中的表达及其对成脂分化的影响。方法 体外贴壁法原代培养的大鼠BMSC细胞经流式细胞术、CD44和CD34免疫荧光染色鉴定后,与椎间盘软骨细胞和成骨细胞一起采用荧光定量RT-PCR方法检测细胞中rno-miR-195-5p的表达情况；采用荧光素酶报告基因实验验证骨形态发生蛋白受体-1α(Bmpr1α)是否为rno-miR-195-5p的靶基因,采用Western印迹法和油红O染色检测rno-miR-195-5p对Bmpr1α表达和BMSC成脂分化的影响。结果流式细胞术与免疫荧光分析显示所培养细胞具备BMSC特征。rno-miR-195-5p在补肾方含药血清干预的原代BMSC中表达明显升高(P<0.01),且在分化完成的软骨细胞和成骨细胞中高表达。双荧光素酶报告实验与Western印迹法证实Bmpr1α是rno-miR-195-5p的靶基因。转染rno-miR-195-5p mimics上调BMSC中rno-miR-195-5p可抑制Bmpr1α的表达(P<0.05),降低成脂分化能力；而转染rno-miR-195-5p inhibitor可显著增加BMSC中Bmpr1α的表达(P<0.01),增强成脂分化能力。结论 补肾方含药血清干预BMSC上调rno-miR-195-5p,通过降低其靶基因Bmpr1α的表达而降低BMSC的成脂分化,改善骨质疏松症状。 |
| 关键词: 骨形态发生蛋白受体-1α rno-miR-195-5p 骨髓间充质干细胞 骨质疏松 |
| DOI：10.16118/j.1008-0392.2017.03.001 |
| 投稿时间：2017-02-07 |
| 基金项目:国家“九七三”重点基础研究发展计划(2013CB531601)；国家自然科学基金(30972633、81173312、81372015)；上海市卫生和计划生育委员会科研项目(201640253)；同济大学附属杨浦医院学科带头人攀登计划(YE2201608) |
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| MicroRNA-195-5p affects adipocyte differentiation in bone mesenchymal stem cells by regulating Bmpr1α gene expression |
| PAN Xin,ZENG Si-liang,LIANG Xing-lun,JI Cheng-dong,LIU Xiao-dong,LIAO Wan-qing |
| (Central Laboratory, Yangpu Hospital, Tongji University, Shanghai 200090, China;Dept.of Clinical Laboratory, Shanghai Junwei Fine Medical Club, Shanghai 200433, China;Dept.of Rehabilitation Therapy, Tianhua College, Shanghai Normal University, Shanghai 201815, China;Dept.of Dermatology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China) |
| Abstract: |
| Objective To investigate the effect of rno-miR-195-5p on modulating adipocyte differentiation related to sensitivity for Bushen decoction-medicated serum of bone marrow mesenchymal stem cells (BMSCs) and its mechanism. Methods BMSCs were isolated and cultured in vitro by adherent culture method. The flow cytometric analysis and immunofluorescent staining were used to identify primarily cultured BMSCs in vitro. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed for verifying the expression of rno-miR-195-5p in primary BMSCs, intervertebral disc chondrocytes and osteoblasts. Bioinformatics was used for predicting target genes for rno-miR-195-5p. Dual-luciferase reporter assay system and Western blot were performed for verifying the target gene and detecting the expression of Bmpr1α. Oil red O staining was used to assess the differentiation of BMSCs to adipocytes by transfection with miRNA mimics or inhibitors. Results The adherent culture method was practicable to obtain a large number of primary rat BMSCs with typical BMSCs characteristics identified by flow cytometry and immunofluorescent staining. After comparing rno-miR-195-5p expression in Bushen decoction-containing serum culture with that from normal saline control culture,the results of RT-PCR showed that rno-miR-195-5p was markedly up-regulated (P<0.01) in BMSCs cultured with Bushen decoction-containing serum. Dual luciferase report assay and Western blotting confirmed that rno-miR-195-5p can affect 3′-UTR sequence in Bmpr1α gene. Bioinformatics result showed that Bmpr1α was the direct target gene for rno-miR-195-5p. Over-expression of rno-miR-195-5p in BMSCs significantly suppressed Bmpr1α expression (P<0.05), and significantly inhibited adipogenic differentiation. We also observed that inhibition of rno-miR-195-5p in BMSCs significantly up-regulated Bmpr1α (P<0.01), and enhanced adipogenic differentiation.ConclusionThe expression of rno-miR-195-5p in rat BMSCs is up-regulated after Bushen decoction-containing serum intervention. rno-miR-195-5p can suppress adipogenic differentiation via regulating Bmpr1α, which may relieve the symptom of osteoporosis. |
| Key words: bone morphogenetic protein receptor type 1A rno-miR-195-5p bone mesenchymal stem cell osteoporosis |