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） 2Da on passively sensitized human airway smooth muscle cells

目的检测1，25-二羟维生素D，[1，25-（OH）2D3]对被动致敏的人气道平滑肌细胞（HASMC）的增殖及其表达基质金属蛋白酶-9（MMP-9）和解整合素-金属蛋白酶33（ADAM33）的影响，探讨其调节支气管哮喘（简称哮喘）患者气道重塑的可能机制。方法用10％哮喘患者血清被动致敏HASMC，以10％非哮喘患者血清为对照。四甲基偶氮唑盐（MTT）法检测不同浓度1，25-（OH）2D3对HASMC细胞增殖活力的变化并确定其有效作用浓度。然后以有效作用浓度的1，25-（OH）2D3预处理HASMC，MTT法测定细胞增殖活力，流式细胞仪测定细胞周期，实时荧光定量PCR及蛋白免疫印迹法分别检测细胞中MMP-9及ADAM33的表达情况。结果（1）1，25．（OH），D，在（10^-9～10^-7）moL／L浓度下能浓度依赖性地抑制被动致敏的HASMC增殖（P〈0．05）；（2）10^-7’mol／L的1，25-（OH）2D3能时间依赖性地抑制被动致敏的HASMC增殖并特异性抑制细胞周期中G1／S的转化；（3）VD组MMP-9及ADAM33蛋白表达较哮喘组分别下降了（63．4±3．6）％和（50．9±2．9）％，但仍显著高于对照组（P〈0．01）；（4）VD组MMP-9及ADAM33mRNA表达较哮喘组分别下降了（52．2±2．5）％和（67．8±3．2）％，但仍显著高于对照组（P〈0．01）。结论1，25-（OH）2D3能从多个层面抑制被动致敏的HASMC的功能，这可能是其调节哮喘气道重塑的作用机制之一。

Objective To investigate the effects of 1,25-（OH） 2D3 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 colorimetri assay was used to examine the effect of 1,25-（ OH）2D3 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 D3 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 ）2D3 was barely detectable at 10^-l0 mol/L. But with the increasing concentration ranging from 10^-9 mol/L to 10^-7 mol/L, l ,25-（OH） 2 D3 markedly inhibited the cell proliferation concentration-dependently and reached the maximum effect at the concentration of 10^-7 mol/L. Accordingly, l0^-7 mol/L was chosen as the optimal concentration of 1,25-（ OH）2D3 for the following study. （2）At the concentration of 10^-7 mol/L, 1,25-（OH） 21）3 inhibited the cell proliferation of passively sensitized HASMCs in a time-dependent manner and hampered the G1/S transition. （3）1,25-（ OH）2D3 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）2D3 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）. Condusion 1,25- （OH）/D3 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 l ,25-（OH） 2D3 in the prevention and therapy of asthmatic airway remodeling.