PTEN在石英诱导的HELF细胞FAK和α-SMA改变中的作用

Role of PTEN in FAK and α-SMA protein levels changes in human embryonic lung fibroblasts induced by silica

目的探讨第10号染色体丢失性磷酸酶-张力蛋白(phosphatase and tensin homolog deleted on chromosome 10,PTEN)在石英诱导人胚肺成纤维细胞(human embryo lung fibroblasts, HELF)黏附斑激酶(focal adhesion kinase, FAK)和平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)改变中的作用。方法分别在含0、25、50、100和200μg/cm2石英的低血清培养基中培养HELF细胞24 h,用细胞计数试剂盒8(cell counting kit-8,CCK8)法确定石英染毒HELF细胞的剂量,同时在倒置显微镜下观察不同剂量石英对HELF细胞形态的影响。50μg/cm^(2)石英溶液分别染毒HELF细胞0、1、2、4、8、12和24 h, 0 h组HELF细胞作为对照组,并用Western blot实验检测HELF在不同染毒时间点的PTEN、p-PTEN、FAK、p-FAK和α-SMA蛋白水平。2×10^(-3) mol/L PTEN抑制剂VO-Ohpic与石英共同处理HELF细胞24 h,同时设空白HELF细胞组和HELF石英染毒组,用Western blot实验检测各组HELF细胞FAK、p-FAK和α-SMA蛋白表达情况。结果随着石英浓度增加,HELF细胞活力呈先增加后下降的变化,其中50μg/cm2石英染毒组细胞活力(144.91±5.10)显著高于0μg/cm2组(101.23±6.57)(P<0.05)。与对照组相比,12 h和24 h的PTEN和p-PTEN表达水平均显著降低[PTEN:12 h为(0.44±0.08),24 h为(0.25±0.02);p-PTEN:12 h为(0.09±0.01),24 h为(0.01±0.00)];而12 h(0.92±0.05)和24 h(0.89±0.01)的FAK,以及24 h时的p-FAK(0.77±0.02)和α-SMA(1.32±0.01)表达水平则显著升高(P<0.05)。石英和PTEN抑制剂共同处理组HELF细胞FAK(0.25±0.03)、p-FAK(0.40±0.02)和α-SMA(0.36±0.01)表达水平均显著高于石英染毒组(P<0.05)。结论在石英诱导HELF细胞FAK、p-FAK和α-SMA表达水平增高的过程中,PTEN负调控FAK、p-FAK和α-SMA表达水平。

OBJECTIVE To investigate the roles of phosphatase and tensin homolog deleted on chromosome 10(PTEN) in focal adhesion kinase(FAK) and α-smooth muscle actin(α-SMA) protein levels changes in human embryonic lung fibroblasts(HELFs) induced by silica. METHODS The HELF cells were cultured in low serum medium containing 0, 25, 50, 100 and 200 μg/cm2 silica for 24 hours, and the cell counting kit-8(CCK8) experiment was used to determine the appropriate dose of silica for stimulation. Meanwhile, the effect of different doses of silica on the morphology of HELFs was observed under inverted microscope. 50 μg/cm^(2) silica solution was used to culture HELFs for 0, 1, 2, 4, 8, 12 and 24 hours(h), and the control group were used as the control group. In addition, Western blot was used to detect HELFs PTEN, p-PTEN, FAK, p-FAK and α-SMA protein levels at each culture time. Besides, HELFs were cultured with 2×10^(-3) mol/L PTEN inhibitor(VO-Ohpic) and/or silica for 24 h, including HELFs group, HELFs plus silica group, and HELFs plus silica plus VO-Ohpic group, and the FAK, p-FAK and α-SMA in each group were also detected by Western blot. RESULTS With the increase of silica dose, HELFs viability firstly increased and then decreased, and the cell viability of 50 μg/cm2 group(144.91±5.10) was significantly higher than that of 0 μg/cm2 group(101.23±6.57)(P<0.05). Compared with the control group of silica treated HELFs, the expression levels of PTEN and p-PTEN at 12 h and 24 h were significantly decreased(PTEN: 0.44±0.08 at 12 h, 0.25±0.02 at 24 h;p-PTEN: 0.09±0.01 at 12 h, 0.01±0.00 at 24 h;all P values<0.05);whereas, FAK at 12 h(0.92±0.05) and 24 h(0.89±0.01), and p-FAK(0.77±0.02) and α-SMA at 24 h(1.32±0.01) were significantly increased(all P values<0.05). The expression levels of FAK(0.25±0.03), p-FAK(0.40±0.02) and α-SMA(0.36±0.01) of HELFs plus silica plus VO-Ohpic group were significantly higher than those of HELFs plus silica group(P<0.05). CONCLUSION While silica induces HELFs FAK, p-FAK, and α-SMA increase, PTEN may downregulate FAK, p-FAK and α-SMA expression levels.