中国组织工程研究

中国组织工程研究 ›› 2017, Vol. 21 ›› Issue (5): 669-675.doi: 10.3969/j.issn.2095-4344.2017.05.003

• 骨髓干细胞 bone marrow stem cells • 上一篇    下一篇

雌激素缺乏型人骨髓间充质干细胞肾虚表型的验证

魏秋实1，2，何  伟1，2，陈镇秋1，陈  达2，洪郭驹2，陈建发1，杨  鹏2   

  1. 1广州中医药大学第一附属医院骨科，广东省广州市  510407；
    2广州中医药大学国家重点学科中医骨伤科学实验室，广东省广州市  510405
  • 出版日期:2017-02-18 发布日期:2017-03-20
  • 通讯作者: 何伟，主任医师，教授，博士生导师，广州中医药大学第一附属医院骨科，广东省广州市 510407；广州中医药大学国家重点学科中医骨伤科实验室，广东省广州市 510405
  • 作者简介:魏秋实，男，1982年生，辽宁省沈阳市人，蒙古族，2012年广州中医药大学毕业，博士，博士后，主要从事股骨头坏死和骨质疏松症发病机制方面的研究。
  • 基金资助:

    国家自然科学基金青年科学基金项目(81302994) 《补肾药通过TAZ与经典Wnt通路crosstalk调控肾虚型骨髓基质干细胞成骨能力的机制》；国家自然科学基金面上项目(81473697)《不同证型股骨头坏死与MRI、病理及分子分型相互关系的探讨研究》； 国家自然科学基金面上项目(81573996) 《microRNA在祛痰逐瘀法促酒精性股骨头坏死骨新生中的调控机制研究》

A preliminary proof of kidney-deficiency phenotype in human bone marrow mesenchymal stem cells with estrogen deficiency

Wei Qiu-shi1, 2, He Wei1, 2, Chen Zhen-qiu1, Chen Da2, Hong Guo-ju2, Chen Jian-fa1, Yang Peng2   

  1. 1Department of Orthopedics Surgery, First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510407, Guangdong Province, China;
    2Key Laboratory of Osteology and Traumatology of TCM, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • Online:2017-02-18 Published:2017-03-20
  • Contact: He Wei, Chief physician, Professor, Doctoral supervisor, Department of Orthopedics Surgery, First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510407, Guangdong Province, China; Key Laboratory of Osteology and Traumatology of TCM, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • About author:Wei Qiu-shi, M.D., Department of Orthopedics Surgery, First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510407, Guangdong Province, China; Key Laboratory of Osteology and Traumatology of TCM, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • Supported by:

    the National Natural Science Foundation of China, No. 81302994, 81473697 and 81573996

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被引次数

2

摘要：

文章快速阅读：

文题释义：
环磷酸腺苷(cAMP)：体内多种激素作用于细胞时，可促使细胞生成环磷酸腺苷，转而调节细胞的生理活动与物质代谢。中医辨证肾虚证与环磷酸腺苷有关，肾阴虚时表现出环磷酸腺苷含量上升，环磷酸腺苷/环磷酸鸟苷比值升高，肾阳虚时表现出环磷酸腺苷含量上升，环磷酸腺苷/环磷酸鸟苷比值降低。实验将环磷酸腺苷作为评价雌激素缺乏型即肾虚型骨髓间充质干细胞表型的鉴定基因。
来曲唑：是新一代芳香化酶抑制剂，通过抑制芳香化酶，使雌激素水平下降，从而消除雌激素对肿瘤生长的刺激作用，用于乳腺癌化疗和放疗。

 

摘要
背景：因雌激素缺乏导致绝经后骨质疏松症患者骨髓间充质干细胞增殖和成骨分化能力下降，从雌激素及其受体角度研究该病的发病机制和治疗策略具有重要意义。
目的：观察雌激素缺乏型骨髓间充质干细胞的肾虚表型特点。
方法：第3代骨髓间充质干细胞在成骨诱导条件下培养，用不同浓度(0，10-10，10-9，10-8，10-7 mol/L)芳香化酶抑制剂来曲唑干预细胞，未诱导的骨髓间充质干细胞作空白对照组。qPCR和Western Blot检测AROM mRNA和蛋白表达，MTT检测细胞增殖能力，碱性磷酸酶活性检测细胞成骨能力，ELISA检测细胞上清液中雌二醇水平，qPCR检测肾虚标志物cAMP mRNA表达。
结果与结论：①10-8 mol/L来曲唑对骨髓间充质干细胞内AROM mRNA和蛋白的抑制效果最好；②培养72 h，10-8 mol/L来曲唑组细胞增殖能力最低；③培养第14天，10-8 mol/L来曲唑组细胞碱性磷酸酶活性低于其他组；④10-8 mol/L来曲唑组细胞上清液中雌二醇水平低于其他组；⑤10-8 mol/L组细胞cAMP mRNA表达低于其他组；⑥结果表明，10-8 mol/L来曲唑在成骨诱导环境中能明显抑制骨髓间充质干细胞增殖及向成骨分化，同时降低骨髓间充质干细胞内cAMP表达及合成和分泌雌二醇的能力，说明适宜浓度来曲唑作为AROM抑制剂建立的雌激素缺乏型骨髓间充质干细胞模型具有肾虚表型特征。

 

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: 0000-0002-1274-9997(魏秋实)

关键词: 干细胞, 骨髓干细胞, 雌激素, 骨髓间充质干细胞, 芳香化酶, 肾虚, 表型, 骨代谢疾病, 国家自然科学基金

Abstract:

BACKGROUND: As the presence of decreased proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs) results from estrogen deficiency-induced postmenopausal osteoporosis, it is of significance to study the relevant pathogenesis and treatment strategies from the perspective of estrogen and its receptors.
OBJECTIVE: To observe the characteristics of kidney-deficiency phenotype in estrogen deficiency-induced BMSCs.
METHODS: Passage 3 BMSCs were cultured in the osteogenic induction medium. The cells were divided into five groups, and cultured with different concentrations (0, 10-10, 10-9, 10-8, 10-7 mol/L) of letrozole. BMSCs with no osteogenic induction were used as blank controls. The expression levels of AROM were detected at mRNA and protein levels by real-time PCR and western blot methods. Cell proliferation of BMSCs was detected using MTT assay. Alkaline phosphatase activity assay was used to detect BMSCs osteogenic ability. ELISA was used to detect estradiol levels in cell supernatants. The expression of cAMP mRNA was detected by real-time PCR method.
RESULTS AND CONCLUSION: 10-8 mol/L Letrozole exhibited the best effect to inhibit the expression of AROM mRNA and protein. After 72 hours of culture, the proliferation of BMSCs fell to the bottom in the 10-8 mol/L letrozole group. After 14 days of culture, the alkaline phosphatase activity in the 10-8 mol/L letrozole group was lower than that in the other groups. ELISA showed that estradiol level in supernatants was lower in the 10-8 mol/L letrozole group than the other groups. Real-time PCR showed that cAMP mRNA expression was lower in the 10-8 mol/L letrozole group than the other groups. These results suggest that 10-8 mol/L letrozole can significantly inhibit the proliferation and osteogenic differentiation of BMSCs, and can significantly decrease the expression of cAMP and the potential of synthesizing and secreting estradiol. Therefore, the process of AROM-mediated estradiol synthesis in BMSCs can be inhibited by 10-8 mol/L letrozole, and has the characteristics of kidney-deficiency phenotype.

 

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

Key words: Bone Marrow, Mesenchymal Stem Cells, Estrogen Antagonists, Tissue Engineering

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