摘要
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.
出处
《中华结核和呼吸杂志》
CAS
CSCD
北大核心
2011年第6期438-441,共4页
Chinese Journal of Tuberculosis and Respiratory Diseases
关键词
哮喘
肌细胞
平滑肌
骨化三醇
细胞增殖
Asthma
Myocytes, smooth muscle
Calcitriol
Cell proliferation