[1]郭　丽,王　娟,周　菁.转化生长因子-β1对人呼吸道平滑肌细胞增殖和迁移的影响[J].新乡医学院学报,2020,37(11):1030-1035.[doi:10.7683/xxyxyxb.2020.11.006]
　GUO Li,WANG Juan,ZHOU Jing.Effect of transforming growth factor-β1 on proliferation and migration of human airway smooth muscle cells[J].Journal of Xinxiang Medical University,2020,37(11):1030-1035.[doi:10.7683/xxyxyxb.2020.11.006]
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转化生长因子-β1对人呼吸道平滑肌细胞增殖和迁移的影响
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《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
37
期数:
2020年11
页码:
1030-1035
栏目:
基础研究
出版日期:
2020-11-05

文章信息/Info

Title:
Effect of transforming growth factor-β1 on proliferation and migration of human airway smooth muscle cells
作者:
郭　丽1; 王　娟1; 周　菁2
（1.空军军医大学第一附属医院老年科，陕西　西安　710054；2.陕西省肿瘤医院肿瘤内科，陕西　西安　710061）
Author(s):
GUO Li1; WANG Juan1; ZHOU Jing2
(1.Department of Cadre Ward,the First Affiliated Hospital of Air Force Medical University,Xi′an 710054,Shaanxi Province,China;2.Department of Oncology,Shaanxi Province Tumor Hospital,Xi′an 710061,Shaanxi Province,China)
关键词:
转化生长因子-β1; 微RNA-21; 人呼吸道平滑肌细胞细胞; 哮喘
Keywords:
transforming growth factor-β1; microRNA-21; human airway smooth muscle cells; asthma
分类号:
R563
DOI:
10.7683/xxyxyxb.2020.11.006
文献标志码:
A
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、10.0 μg·L-1TGF-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10 μg·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<0.05），中剂量TGF-β1组、高剂量TGF-β1组细胞划痕愈合率显著高于低剂量TGF-β1组（P<0.05），高剂量TGF-β1组细胞划痕愈合率显著高于中剂量TGF-β1组（P<0.05）；miR-21 inhibitor组细胞划痕愈合率显著低于对照组和阴性对照组（P<0.05），TGF-β1＋miR-21 inhibitor组细胞划痕愈合率显著高于miR-21 inhibitor组（P<0.05）。低剂量TGF-β1组、中剂量TGF-β1组、高剂量TGF-β1组细胞中miR-21相对表达量显著高于空白组（P<0.05）；中剂量TGF-β1组、高剂量TGF-β1组细胞中miR-21相对表达量显著高于低剂量TGF-β1组（P<0.05）；高剂量TGF-β1组细胞中miR-21相对相对表达量显著高于中剂量TGF-β1组（P<0.05）。与对照组比较，高剂量TGF-β1组细胞PTEN相对表达量降低，p-Akt/Akt水平提高（P<0.05）。与对照组比较，miR-21 inhibitor组细胞PTEN相对表达量升高，p-Akt/Akt 水平下降（P<0.05）。与miR-21 inhibitor组比较，TGF-β1＋miR-21 inhibitor组PTEN相对表达量降低，p-Akt/Akt水平升高（P<0.05）。结论　TGF-β1可促进HASM细胞的增殖和迁移，且呈剂量依赖性，其作用机制可能与上调miR-21表达及激活PTEN/Akt信号通路有关。
Abstract:
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 10 μg·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 10 μg·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- dose TGF-β1 groups were significantly higher than that in the blank group (P<0.05),the proliferation ability of cells in the medium- and high- dose TGF-β1 groups were significantly higher than that in the low-dose TGF-β1 group (P<0.05),and the proliferation ability of cells in the high-dose TGF-β1 group were significantly higher than that in the medium-dose TGF-β1 group (P<0.05).The proliferation ability of cells in the miR-21 inhibitor group was significantly lower than that in the negative control group and the control group (P<0.05),and the proliferation ability in the TGF-β1+ miR-21 inhibitor group was significantly higher than that in the miR-21 inhibitor group (P<0.05).After 48 h of cell culture,the cell healing rates in the low-,medium- and high-dose TGF-β1 groups were significantly higher than that in the blank group (P<0.05),the cell healing rates in the medium- and high-dose TGF-β1 groups were significantly higher than that in the low-dose group (P<0.05),and the cell healing rate in the high-dose TGF-β1 group was significantly higher than that in the medium-dose TGF-β1 group,the differences were statistically signifcant (P<0.05).The cell healing rate in miR-21 inhibitor group was significantly lower than that in the control group and negative control group (P<0.05),and the cell healing rate in the TGF-β1+ miR-21 inhibitor group was significantly higher than that in the miR-21 inhibitor group (P<0.05).The relative expression level of miR-21 in the low-,medium-,and high-dose TGF-β1 groups was significantly higher than that in the blank group (P<0.05).The relative expression level of miR-21 in the cells of the medium- and high- dose TGF-β1 groups was significantly higher than that in the low-dose TGF-β1 group (P<0.05).The relative expression level of miR-21 in the high-dose TGF-β1 group was significantly higher than that in the medium-dose TGF-β1 group (P<0.05).Compared with the control group,the relative expression level of PTEN decreased and p-Akt/Akt level increased in the high-dose TGF-β1 group (P<0.05).Conclusion　TGF-β1 can promote the proliferation and migration of HASM cells in a dose-dependent manner,and its mechanism may be related to the up-regulation of miR-21 expression and the activation of PTEkt signaling pathway.

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更新日期/Last Update: 2020-11-05