[1,25-二羟维生素D3对被动致敏人气道平滑肌细胞的抑制作用]

[Inhibition of 1,25-(OH)2D3 on passively sensitized human airway smooth muscle cells].

作者信息

Song Ying-fang, Lai Guo-xiang, Qi Hao-wen, Wu Chang-gui

机构信息

Division of Pulmonary and Critical Care Medicine, Fuzhou General Hospital, Nanjing Military Command, Fuzhou 350025, China.

出版信息

Zhonghua Jie He He Hu Xi Za Zhi. 2011 Jun;34(6):438-41.

PMID:21781516

Abstract

OBJECTIVE

To investigate the effects of 1,25-(OH)(2)D(3) on the proliferation of passively sensitized human airway smooth muscle cells (HASMCs) and their expressions of MMP-9 and a disintegrin and metalloprotease 33(ADAM33).

METHODS

HASMCs were passively sensitized with 10% serum from asthmatic patients. MTT colorimetry assay was used to examine the effect of 1,25-(OH)(2)D(3) on cell proliferation at different concentrations (10(-10) mol/L, 10(-9) mol/L, 10(-8) mol/L, 10(-7) mol/L).By this way, its optimal inhibitory concentration was determined. And then the effects of 1,25-(OH)(2)D(3) at the optimal concentration on cell proliferation was examined by the same MTT assay and cell cycle analysis by flow cytometry. The expressions of MMP-9 and ADAM33 in HASMCs were studied by real-time quantitative RT-PCR and Western blotting analysis.

RESULTS

(1) Inhibition of cell proliferation by 1,25-(OH)(2)D(3) was barely detectable at 10(-10) mol/L. But with the increasing concentration ranging from 10(-9) mol/L to 10(-7) mol/L, 1,25-(OH)(2)D(3) markedly inhibited the cell proliferation concentration-dependently and reached the maximum effect at the concentration of 10(-7) mol/L. Accordingly, 10(-7) mol/L was chosen as the optimal concentration of 1,25-(OH)(2)D(3) for the following study. (2) At the concentration of 10(-7) mol/L, 1,25-(OH)(2)D(3) inhibited the cell proliferation of passively sensitized HASMCs in a time-dependent manner and hampered the G(1)/S transition. (3) 1,25-(OH)(2)D(3) pretreatment attenuated the MMP-9 and ADAM33 protein levels in passively sensitized HASMCs by (63.4 ± 3.6)% and (50.9 ± 2.9)%, respectively (P < 0.01). (4) 1,25-(OH)(2)D(3) significantly inhibited the MMP-9 and ADAM33 mRNA levels in passively sensitized HASMCs by (52.2 ± 2.5)% and (67.8 ± 3.2)%, respectively (P < 0.01).

CONCLUSION

1,25-(OH)(2)D(3) has a direct inhibitory effect on passively sensitized HASMCs in vitro, including the inhibition of cell proliferation and the expressions of MMP-9 and ADAM33, which maybe associated with the beneficial role of 1,25-(OH)(2)D(3) in the prevention and therapy of asthmatic airway remodeling.

摘要

目的

探讨1,25 -（OH）₂D₃对被动致敏的人气道平滑肌细胞（HASMCs）增殖及其基质金属蛋白酶-9（MMP - 9）和去整合素金属蛋白酶33（ADAM33）表达的影响。

方法

用哮喘患者的10%血清对HASMCs进行被动致敏。采用MTT比色法检测不同浓度（10⁻¹⁰mol/L、10⁻⁹mol/L、10⁻⁸mol/L、10⁻⁷mol/L）的1,25 -（OH）₂D₃对细胞增殖的影响，以此确定其最佳抑制浓度。然后用相同的MTT法和流式细胞术进行细胞周期分析，检测最佳浓度的1,25 -（OH）₂D₃对细胞增殖的影响。通过实时定量逆转录-聚合酶链反应（RT - PCR）和蛋白质印迹分析研究HASMCs中MMP - 9和ADAM33的表达。

结果

（1）10⁻¹⁰mol/L的1,25 -（OH）₂D₃对细胞增殖的抑制作用几乎检测不到。但随着浓度从10⁻⁹mol/L增加到10⁻⁷mol/L，1,25 -（OH）₂D₃明显呈浓度依赖性抑制细胞增殖，并在10⁻⁷mol/L浓度时达到最大效应。因此，选择10⁻⁷mol/L作为1,25 -（OH）₂D₃在后续研究中的最佳浓度。（2）在10⁻⁷mol/L浓度时，1,25 -（OH）₂D₃以时间依赖性方式抑制被动致敏的HASMCs的细胞增殖，并阻碍G₁/S期转换。（3）1,25 -（OH）₂D₃预处理使被动致敏的HASMCs中MMP - 9和ADAM33蛋白水平分别降低（63.4±3.6）%和（50.9±2.9）%（P<0.01）。（4）1,25 -（OH）₂D₃显著抑制被动致敏的HASMCs中MMP - 9和ADAM33 mRNA水平，分别降低（52.2±2.5）%和（67.8±3.2）%（P<0.01）。