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

doi:10.3969/j.issn.2095-4344.2017.05.003

Abstract

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

CLC Number:

R394.2

Wei Qiu-shi, He Wei, Chen Zhen-qiu, Chen Da, Hong Guo-ju, Chen Jian-fa, Yang Peng. A preliminary proof of kidney-deficiency phenotype in human bone marrow mesenchymal stem cells with estrogen deficiency[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(5): 669-675.

Figures/Tables 2

2.1 骨髓间充质干细胞表面标记表达细胞培养至第2代，细胞表面标记CD73(99.9%)、CD44(100%)、CD29 (99.0%)阳性，CD11b(0.3%)、CD45(0.4%)、HLA-DR(0.4%)阴性，表明培养细胞为骨髓间充质干细胞(图1)。 2.2 碱性磷酸酶染色、茜素红S染色检测骨髓间充质干细胞成骨分化能力骨髓间充质干细胞培养第14天的碱性磷酸酶染色结果及培养至第21天的茜素红染色结果定量分析，诱导组骨髓间充质干细胞显染程度高于单纯培养组，差异有显著性意义，显示诱导组骨髓间充质干细胞表现出更高的成骨细胞转化率和成骨分化能力(图2)。 2.3 MTT检测骨髓间充质干细胞的增殖能力 10-8 mol/L来曲唑组细胞增殖能力明显低于正常对照组、0、10-10、10-9 mol/L来曲唑组(均为P < 0.001)，与10-7 mol/L来曲唑组比较差异无显著性意义(图3)。 2.4 碱性磷酸酶活性检测骨髓间充质干细胞的成骨能力细胞培养第14天，10-8 mol/L来曲唑组细胞碱性磷酸酶活性明显低于正常对照组、0、10-10、10-9 mol/L来曲唑组(均为P < 0.001)，与10-7 mol/L来曲唑组比较差异无显著性意义(图4)。 2.5 qPCR和Western blot检测AROM的表达水平用不同浓度(0，10-10，10-9，10-8，10-7 mol/L)芳香化酶抑制剂来曲唑干预经成骨诱导液预处理的细胞，10-8 mol/L来曲唑组AROM mRNA表达明显低于空白对照组、0、10-10、10-9 mol/L来曲唑组(均为P < 0.001，图5)，尽管与10-7 mol/L来曲唑组比较差异无显著性意义，但其相对表达量亦低于后者。10-8 mol/L来曲唑组AROM蛋白表达明显低于空白组、0、10-10、10-9、10-7 mol/L来曲唑组(均为P < 0.05，图6)，说明10-8 mol/L来曲唑在mRNA和蛋白2个层面对AROM抑制效果最强。 2.6 ELISA检测细胞上清液雌二醇水平 10-8 mol/L来曲唑组细胞上清液中雌二醇水平明显低于空白对照组、0、10-10、10-9、10-7 mol/L来曲唑组(均为P < 0.01，图7)。 2.7 qPCR检测cAMP的表达水平 10-8 mol/L来曲唑组cAMP mRNA表达明显低于0、10-10、10-9、10-7 mol/L来曲唑组(均为P < 0.001，图8)，与空白对照组比较差异无显著性意义。"

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