中国组织工程研究

中国组织工程研究 ›› 2019, Vol. 23 ›› Issue (21): 3281-3288.doi: 10.3969/j.issn.2095-4344.1742

• 骨髓干细胞 bone marrow stem cells •    下一篇

低氧诱导c-Kit+骨髓间充质干细胞增殖及旁分泌中ROCK信号的作用

邵钟铭，王可可，廖晓敏，哈艳平，李汝佳，申志华，揭 伟   

  1. 广东医科大学基础医学院病理学系，广东省湛江市 524023
  • 修回日期:2019-01-21 出版日期:2019-07-28 发布日期:2019-07-28
  • 通讯作者: 揭伟，博士，教授，广东医科大学基础医学院病理学系，广东省湛江市 524023
  • 作者简介:邵钟铭，女，1992年生，江苏省盐城市人，汉族，广东医科大学在读硕士，主要从事心血管病理研究。 并列第一作者：王可可，女，1991年生，安徽省蚌埠市人，汉族，四川大学在读博士学位，主要从事心衰与逆转病理生理方面的研究。
  • 基金资助:

    国家自然科学基金项目(81670254)，项目负责人：揭伟；广东省科技计划项目(2016A020214016)，项目负责人：揭伟；广东省“扬帆计划”高层次人才项目(4YF16007G)，项目负责人：揭伟

Roles of ROCK signaling in proliferation and paracrine action of hypoxia-induced c-Kit+ bone marrow mesenchymal stem cells

Shao Zhongming, Wang Keke, Liao Xiaomin, Ha Yanping, Li Rujia, Shen Zhihua, Jie Wei   

  1. Department of Pathology, School of Basic Medicine, Guangdong Medical University, Zhanjiang 524023, Guangdong Province, China
  • Revised:2019-01-21 Online:2019-07-28 Published:2019-07-28
  • Contact: Jie Wei, PhD, Professor, Department of Pathology, School of Basic Medicine, Guangdong Medical University, Zhanjiang 524023, Guangdong Province, China
  • About author:Shao Zhongming, Master candidate, Department of Pathology, School of Basic Medicine, Guangdong Medical University, Zhanjiang 524023, Guangdong Province, China. Wang Keke, Doctorate candidate, Department of Pathology, School of Basic Medicine, Guangdong Medical University, Zhanjiang 524023, Guangdong Province, China. Shao Zhongming and Wang Keke contributed equally to this work.
  • Supported by:

    the National Natural Science Foundation of China, No. 81670254 (to JW); the Science and Technology Planning Project of Guangdong Province, No. 2016A020214016 (to JW); and the YangFan Plan of Guangdong Province, No. 4YF16007G (to JW)

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摘要：

文章快速阅读：

文题释义：
ROCK信号：ROCK属于丝氨酸/苏氨酸激酶，是Rho激酶的下游靶分子，组成Rho/ROCK信号。ROCK信号通过磷酸化下游的效应蛋白(MLC、Lin-11、Isl-1、LIMK、ERM、MARCKS、CRMP-2等)来实现其生物学功能，具有调节细胞收缩、迁移、黏附、增殖及分化等作用，其异常活化与心血管疾病、神经系统疾病、肿瘤等密切相关。ROCK分为ROCK1与ROCK2两种亚型，两者氨基酸序列一致性为65%，但激酶结构域高度保守。ROCK1与ROCK2在机体分布上具有显著差异性。ROCK信号的活化可以通过下游分子如p-MLC2和MLC2表达比值的变化来判断。
旁分泌：细胞分泌某种细胞因子、激素或小分子活性物质作用于相邻细胞并产生生物学效应。旁分泌是干细胞治疗组织损伤修复相关转化医学中重要的机制之一。

 

摘要
背景：研究显示低氧能刺激骨髓间充质干细胞增殖，但具体机制尚不清楚。ROCK信号对细胞增殖、凋亡等具有重要影响，当前对ROCK信号与骨髓间充质干细胞的生物学功能研究较少。  
目的：分析ROCK信号在低氧诱导c-Kit+骨髓间充质干细胞增殖及旁分泌中的作用。
方法：贴壁法获得大鼠股骨骨髓间充质干细胞，应用磁激活细胞分选法获得c-Kit+亚群，细胞培养于常氧(体积分数21%O2)及低氧(体积分数2%O2)条件下，并用ROCK信号抑制剂Fasudil (10 μmol/L)处理细胞。观察：①Western blot检测p-MLC2(T18/S12)和MLC2蛋白表达，以p-MLC2(T18/S12)/MLC2比值变化反映ROCK信号的活化情况；②细胞计数分析细胞增殖，Western blot检测细胞增殖标记物PCNA和细胞周期相关蛋白的表达；③流式细胞术检测细胞周期的变化；④ELISA检测细胞培养上清中血管内皮生长因子、转化生长因子β1和碱性成纤维细胞生长因子水平，了解细胞的旁分泌情况。
结果与结论：成功分选出大鼠c-Kit+骨髓间充质干细胞。①相对于常氧而言，低氧显著下调MLC表达和上调p-MLC(T18/S19)表达进而增加p-MLC(T18/S19)/MLC2比值；②低氧促进c-Kit+骨髓间充质干细胞的增殖，表现为细胞数量增多和PCNA蛋白表达上调，Fasudil减弱了低氧诱导的c-Kit+骨髓间充质干细胞增殖，使细胞周期阻滞于S期并抑制CDK2、CDK4和上调p16的表达；③低氧显著促进c-Kit+骨髓间充质干细胞分泌上述3种细胞因子，Fasudil减弱低氧诱导的血管内皮生长因子和转化生长因子β1但上调碱性成纤维细胞生长因子的分泌。实验结果提示低氧刺激促进c-Kit+骨髓间充质干细胞增殖和旁分泌并活化ROCK信号；Fasudil抑制低氧诱导的细胞增殖和旁分泌作用，提示ROCK信号对c-Kit+骨髓间充质干细胞的增殖和旁分泌具有重要的正性调节作用。


中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程
ORCID: 0000-0002-1869-1458(揭伟)

关键词: ROCK信号, Fasudil, 低氧, 干细胞, 骨髓间充质干细胞, c-Kit, 细胞增殖, 旁分泌, 国家自然科学基金

Abstract:

BACKGROUND: Hypoxia stimulates the proliferation of bone marrow mesenchymal stem cells, but the specific mechanism is still unclear. The Rho-associated coiled-coil containing kinases (ROCK) signaling has an important influence on cell proliferation, migration and apoptosis. Few studies have been reported on the effect of ROCK signaling on the biological functions of bone marrow mesenchymal stem cells.
OBJECTIVE: To analyze the effect of ROCK signaling on the hypoxia-induced proliferation and paracrine of c-Kit+ bone marrow mesenchymal stem cells.
METHODS: Rat bone marrow mesenchymal stem cells derived from the rat femur were obtained by adherent method, and the c-Kit+ subpopulation was sorted by magnetic activated cell sorting. Cells were cultured under normoxia (21% O2) and hypoxia (2% O2). After treatment with an ROCK signal inhibitor Fasudil (10 μmol/L), western blot was used to detect the expression of p-MLC (T18/S19), MLC, p-MLC2(T18/S12)/MLC2, PCNA and cell cycle related proteins. The cell proliferation was analyzed by cell counting, the cell cycle was detected using flow cytometry, and the changes of vascular endothelial growth factor, transforming growth factor β1 and basic fibroblast growth factor in the cell supernatant were detected by ELISA. 
RESULTS AND CONCLUSION: c-Kit+ subpopulation were successfully isolated from rat bone marrow mesenchymal stem cells. Compared with normoxia, hypoxia treatment significantly down-regulated MLC expression, up-regulated p-MLC(T18/S19) expression and therefore increased the ratio of p-MLC(T18/S19)/MLC2. Meanwhile, hypoxia promoted the proliferation of c-Kit+ bone marrow mesenchymal stem cells, as evidenced by the increase in cell number and PCNA protein expression. Fasudil inhibited the hypoxia-induced the proliferation of c-Kit+ bone marrow mesenchymal stem cells, arrested the cell cycle at S phase, inhibited CDK2 and CDK4 and increased p16 expression. Hypoxia significantly promoted the secretion of vascular endothelial growth factor, transforming growth factor β1 and basic fibroblast growth factor by c-Kit+ bone marrow mesenchymal stem cells, and Fasudil attenuated hypoxia-induced vascular endothelial growth factor and transforming growth factor β1 expression but astonishingly upregulated basic fibroblast growth factor level. These results indicate that hypoxia promotes cell proliferation and paracrine of angiogenic factors and activates ROCK signaling in c-Kit+ bone marrow mesenchymal stem cells; Fasudil inhibits hypoxia-induced proliferation and expression of angiogenic factors in c-Kit+ bone marrow mesenchymal stem cells, suggesting that ROCK signaling plays a positive role in the proliferation and paracrine of c-Kit+ bone marrow mesenchymal stem cells.

Key words: ROCK signal, Fasudil, hypoxia, stem cells, bone marrow mesenchymal stem cells, c-Kit, cell proliferation, paracrine, National Natural Science Foundation of China

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