目的 观察1,25-(OH)2D3对哮喘大鼠AMSCs增殖及RhoA表达的影响,探讨其在哮喘治疗中的作用.方法 用卵白蛋白致敏和激发建立急性哮喘模型,原代培养急性哮喘大鼠的ASMCs,以1,25-(OH)2D3作为干预因素,CCK8法检测1,25-(OH)2D3对ASMCs增殖的影响,测定细胞增殖活力;用TNF-α、1,25-(OH)2D3和地塞米松(DXM)处理体外培养的急性哮喘大鼠ASMCs并分组:对照组(N)、哮喘组(A组)、1,25-(OH)2D3组(VD组)、DXM组、1,25-(OH) 2D3 +DXM联合治疗组(L组).用Transwell小室检测细胞迁移;流式细胞仪测定细胞周期.同时采用Real time PCR和Western blot方法检测肺组织和ASMCs中RhoA的表达变化,研究其作用机制.结果 1,25-(OH)2D3在10-9～ 10-6mol/L浓度下能显著抑制ASMCs的增殖,且为浓度依赖性(P＜0.05);1,25-(OH) 2D3对哮喘大鼠的ASMCs的抗增殖作用呈现时间依赖性;1,25-(OH) 2D3对哮喘大鼠的ASMCs迁移有明显的抑制作用;1,25-(OH) 2D3显著抑制哮喘大鼠ASMCs细胞周期中G1/S期的转化;1,25-(OH)2D3抑制并减少了RhoA/Rho激酶信号通路中RhoA基因和蛋白的表达.结论 1,25-(OH)2D3能显著抑制哮喘大鼠ASMCs的增殖、迁移及细胞周期的进展,这种多重的抑制作用可能是其通过调控RhoA/Rho激酶信号通路而实现其调节哮喘气道炎症、AHR和气道重塑的作用机制之一.%Objective To observe the effect of 1,25-(OH)2 D3 on proliferation of ASMCs and RhoA in asthma rats,and explore its role in the treatment of asthma.Methods Acute asthma models of Wistar rats were established with ovalbumin sensitizing and challenging,ASMCs of acute asthmatic rats were primarily cultured.1,25-(OH)2D3 as the intervention factors,the effect of 1,25-(OH)2D3 on proliferation and proliferation activity of ASMCs was determined by CCK8.The cultured ASMCs of acute asthma rat in vitro were divided into 5 groups:control group (group N),TNF-αt group (group A),1,25-(OH) 2 D3 group (group VD),DXM group,1,25-(OH) 2D3 + DXM group (group L).The cell migration was detected by Transwell chamber;cell cycle was analyzed by flow cytometry.Meanwhile,the expression of RhoA in lung tissue and ASMCs was detected by Real time PCR and Western blot,and the mechanism of action was analyzed.Results 1,25-(OH) 2D3 could significantly inhibit the proliferation of ASMCs in a concentration dependent manner (P ＜ 0.05);1,25-(OH)2D3 had anti-proliferative effects on ASMCs of asthmatic rats in a time dependent manner;1,25-(OH) 2 D3 had obviously inhibitory effect on the migration of ASMCs in asthmatic rats;1,25-(OH)2D3 could significantly inhibit the cell cycle conversion during G1/S period in ASMCs of asthma rats;1,25-(OH)2D3 could inhibit and decrease the expression of RhoA gene and protein in Rho/Rho kinase signaling pathway.Conclusion 1,25-(OH) 2D3 can inhibit the proliferation,migration and cell cycle progression of ASMCs,which may be one of the mechanisms by which RhoA/Rho kinase signaling pathway regulates airway inflammation,AHR and airway remodeling in asthma.
展开▼
