雌激素缺乏型人骨髓间充质干细胞肾虚表型的验证

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

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。q PCR和Western Blot检测AROM mR NA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,qP CR检测肾虚标志物cAMP mRNA表达。结果与结论:(1)10^(-8) mol/L来曲唑对骨髓间充质干细胞内AROM mR NA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;(4)10^(-8) mol/L来曲唑组细胞上清液中雌二醇水平低于其他组;(5)10^(-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。

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^-7mol/L） of letrozole. BMSCs with no osteogenic induction were used as blank controls. The expression levels of AROM were detected at m RNA 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 c AMP m RNA 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 m RNA 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 c AMP m RNA 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 c AMP 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.