摘要
目的探究转化生长因子-β1(TGF-β1)对人呼吸道平滑肌(HASM)细胞增殖、迁移的影响。方法采集于空军军医大学第一附属医院行肺叶切除术的2例哮喘患者的肺叶标本并获取HASM细胞。将处于对数生长期的HASM细胞随机分为空白组、低剂量TGF-β1组、中剂量TGF-β1组和高剂量TGF-β1组,低剂量TGF-β1组、中剂量TGF-β1组和高剂量TGF-β1组细胞分别应用终质量浓度为0.1、1.0、10.0μg·L-1 TGF-β1进行处理,空白组细胞应用等量磷酸盐缓冲液处理。另将HASM细胞随机分为对照组、阴性对照组、微RNA-21(miR-21)inhibitor组和TGF-β1+miR-21 inhibitor组。对照组细胞不作任何处理,阴性对照组细胞转染阴性对照载体,miR-21 inhibitor组细胞转染miR-21 inhibitor,TGF-β1+miR-21 inhibitor组细胞转染miR-21 inhibitor后以10μg·L-1 TGF-β1处理48 h。应用细胞计数试剂盒-8和划痕实验分别检测各组细胞增殖和迁移情况,采用实时荧光定量聚合酶链式反应检测空白组、低剂量TGF-β1组、中剂量TGF-β1组、高剂量TGF-β1组细胞miR-21相对表达量,采用蛋白免疫印迹法检测对照组、高剂量TGF-β1组、miR-21 inhibitor组、TGF-β1+miR-21 inhibitor组细胞第10号染色体缺失的磷酸酶及张力蛋白同源等位基因(PTEN)、蛋白激酶B(Akt)、磷酸化Akt(p-Akt)蛋白相对表达量。结果各组细胞铺板培养至12、24、48 h时,低剂量TGF-β1组、中剂量TGF-β1组、高剂量TGF-β1组细胞增殖能力显著高于空白组(P<0.05),中剂量TGF-β1组、高剂量TGF-β1组细胞增殖能力显著高于低剂量TGF-β1组(P<0.05),高剂量TGF-β1组细胞增殖能力显著高于中剂量TGF-β1组(P<0.05);miR-21 inhibitor组细胞增殖能力显著低于阴性对照组和对照组(P<0.05),TGF-β1+miR-21 inhibitor组细胞增殖能力显著高于miR-21 inhibitor组(P<0.05)。细胞划痕培养48 h后,低剂量TGF-β1组、中剂量TGF-β1组、高剂量TGF-β1组细胞划痕愈合率显著高于空白组(P<0.0
Objective To investigate the effect of transforming growth factor-β1(TGF-β1)on the proliferation and migration of human airway smooth muscle(HASM)cells.Methods Lung lobe specimens were obtained from 2 patients with asthma who underwent lobectomy in the First Affiliated Hospital of Air Force Military Medical University,and HASM cells were obtained.HASM cells in logarithmic growth phase were randomly divided into the blank group,low-dose TGF-β1 group,medium-dose TGF-β1 group,and high-dose TGF-β1 group,the cells in the low-dose TGF-β1 group,medium-dose TGF-β1 group and high-dose TGF-β1 group were treated with TGF-β1 at different final mass concentrations of 0.1,1.0 and 10μg·L-1,respectively;the cells in the blank group were treated with equal volume of phosphate buffered solution.In addition,HASM cells were randomly divided into the control group,negative control group,microRNA-21(miR-21)inhibitor group and TGF-β1+miR-21 inhibitor group.The cells in the control group were not treated,the cells in the negative control group were transfected with the negative control vector,the cells in the miR-21 inhibitor group were transfected with miR-21 inhibitor,and the cells in the TGF-β1+miR-21 inhibitor group were treated with 10μg·L-1 TGF-β1 for 48 h after transfection with miR-21 inhibitor.Cell counting kit-8 and scratch assay were used to detect the cell proliferation and migration in each group.The relative expression levels of miR-21 in the blank group,low-dose TGF-β1 group,medium-dose TGF-β1 group and high-dose TGF-β1 group were measured by real-time fluorescence quantitative polymerase chain reaction.The relative expression levels of phosphatase and tensin homolog deleted on chromosome ten(PTEN),protein kinase B(Akt)and phosphorylated Akt(p-Akt)of the cells in the control group,high-dose TGF-β1 group,miR-21 inhibitor group,TGF-β1+miR-21 inhibitor group were detected by Western blot.Results When the cells were cultured for 12,24 and 48 h,the proliferation ability of cells in the low-,medium-and high
作者
郭丽
王娟
周菁
GUO Li;WANG Juan;ZHOU Jing(Department of Cadre Ward,the First Affiliated Hospital of Air Force Medical University,Xi′an 710054,Shaanxi Province,China;Department of Oncology,Shaanxi Province Tumor Hospital,Xi′an 710061,Shaanxi Province,China)
出处
《新乡医学院学报》
CAS
2020年第11期1030-1035,共6页
Journal of Xinxiang Medical University
