17β-雌二醇对大鼠骨髓间充质干细胞成骨分化过程中Ca2+通道的作用

doi:10.13481/j.1671-587x.20190512

摘要/Abstract

摘要： 目的：观察17β-雌二醇（17β-E2）对大鼠骨髓间充质干细胞（MSCs）成骨分化过程中钙离子（Ca²⁺）通道的影响，阐明17β-E2对MSCs促成骨分化的作用机制。方法：采用密度梯度离心法和贴壁筛选法分离出MSCs，连续传代3次，进行成骨细胞的诱导分化。将MSCs分为对照组[单纯成骨细胞培养基（OBM）培养]和不同剂量17β-E2组（在OBM中分别添加0.1、1.0、10.0和100.0 pmol·L^-1 17β-E2）。成骨诱导14 d，各组细胞经Fluo-3/AM染色后，利用激光扫描共聚焦显微镜测定平均荧光强度（MFI），以MFI代表Ca²⁺水平。应用全细胞膜片钳技术记录不同条件下的全细胞Ca²⁺电流。结果：采用密度梯度离心法和贴壁筛选法成功分离MSCs。MSCs细胞呈成纤维细胞样，核呈椭圆形，细胞核内可见核仁。传代培养的MSCs生长旺盛，保持原代细胞的形态特征。随着17β-E2浓度的增加，各组细胞的Ca²⁺水平也逐渐增强，以100.0 pmol·L^-1 17β-E2组最为明显。与对照组比较，10.0和100.0 pmol·L^-1 17β-E2组的MSCs成骨分化过程中Ca²⁺水平和Ca²⁺电流峰值明显升高（P<0.05或P<0.01），0.1和1.0 pmol·L^-1 17β-E2组Ca²⁺水平和Ca²⁺电流峰值差异无统计学意义（P>0.05）。结论：密度梯度离心法和贴壁筛选法分离所得细胞为大鼠MSCs。17β-E2通过增强MSCs Ca²⁺通道的开放，增强Ca²⁺内向电流发挥促进成骨形成作用，并呈剂量依赖性。

关键词: 骨髓间充质干细胞, 17β-雌二醇, 钙离子通道, 成骨细胞

Abstract: Objective:To observe the effect of 17β-estradiol (17β-E2) on the calcium (Ca²⁺) channels during the osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs), and to elucidate the mechanism of 17β-E2 in the osteogenic differentiation of MSCs. Methods:The MSCs were separated by density gradient centrifugation and adherent screening, and passaged for 3 times continuously to induce osteoblast differentiation. The MSCs were divided into control group[cultivated in osteoblast culture medium alone (OBM)] and different doses of 17β-E2 groups(added with 0.1, 1.0, 10.0, and 100.0 pmol·L^-1 17β-E2 in OBM, respectively). On the 14th day of osteogenic induction, the cells in each group were stained with Fluo-3/AM, and the Ca²⁺ levels were determinated by laser scanning confocal microscope;the mean fluorescence intensity (MFI) was used to respresent the level of Ca²⁺. Whole-cell Ca²⁺ currents were recorded using whole-cell patch clamp technique under different conditions. Results:The MSCs with fibroblast-like cells, oval nuclei and visible nucleoli were successfully isolated by density gradient centrifugation and adherent screening. The subcultured MSCs grew vigorously and maintained the morphological characteristics of primary cells. Following the increase of 17β-E2 concentration,the Fluo-3 fluorescence staining intensity of Ca²⁺ in each group was also gradually increased, especially in 100.0 pmol·L^-1 17β-E2 group. Compared with control group, the MFI of Ca²⁺ and the current peak values of Ca²⁺ in 10.0 and 100.0 pmol·L^-1 17β-E2 groups were increased (P<0.05 or P<0.01) during osteogenic differentiation of the MSCs; the MFI of Ca²⁺ and the current peak values of Ca²⁺ in 0.1 and 1.0 pmol·L^-1 17β-E2 groups showed no significant differences (P>0.05). Conclusion:The cells isolated by density gradient and adherent screening method are the rat MScs. 17β-E2 plays a role in promoting osteogenesis by enhancing the opening of Ca²⁺ channels in the MSCs and the inward current of calcium ions in a dose-dependent manner.

Key words: mesenchymal stem cells, 17β-estradiol, calcium channels, osteoblasts

中图分类号:

Q813

冷冰, 王大麟, 郑淑云, 韩梅, 吴震宇, 裴颖, 薛昊罡. 17β-雌二醇对大鼠骨髓间充质干细胞成骨分化过程中Ca²⁺通道的作用[J]. 吉林大学学报(医学版), 2019, 45(05): 1041-1045.

LENG Bing, WANG Dalin, ZHENG Shuyun, HAN Mei, WU Zhenyu, PEI Ying, XUE Haogang. Effect of 17β-estradiol on Ca²⁺ channels during osteogenic differentiation of rat bone marrow mesenchymal stem cells[J]. Journal of Jilin University(Medicine Edition), 2019, 45(05): 1041-1045.

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17β-雌二醇对大鼠骨髓间充质干细胞成骨分化过程中Ca²⁺通道的作用

Effect of 17β-estradiol on Ca²⁺ channels during osteogenic differentiation of rat bone marrow mesenchymal stem cells

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