抑制Akt信号通路对病理性瘢痕成纤维细胞生长、细胞周期及凋亡的影响
Effects of inhibiting Akt signaling pathway on growth, cell cycle and apoptosis of fibroblasts in pathological scar

目的:研究抑制蛋白激酶B(Akt)信号通路对病理性瘢痕成纤维细胞生长、细胞周期及凋亡的影响。方法:体外分离培养人病理性瘢痕成纤维细胞,用不同浓度的Akt信号通路抑制剂LY294002处理后,采用MTT法检测细胞增殖情况,计算IC50。用IC50浓度的LY294002处理病理性瘢痕成纤维细胞后,流式细胞术检测细胞周期和凋亡情况,Western blot检测活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(Cleaved Caspase-9)、Akt、磷酸化的Akt(p-Akt)水平。结果:不同浓度的LY294002作用后细胞在570 nm处的吸光度值(A值)降低(P<0.001),IC50为30 mg/L。30 mg/L LY294002作用后细胞被阻滞在G0/G1期(P<0.001)。30 mg/L LY294002作用后细胞凋亡率高于0 mg/L作用组,细胞中Cleaved Caspase-3、Cleaved Caspase-9表达水平高于0 mg/L作用组,而p-Akt表达水平明显低于0 mg/L作用组(P<0.001)。结论:抑制Akt信号通路能够抑制病理性瘢痕成纤维细胞生长,阻滞细胞周期,促进细胞凋亡,促进细胞中Caspase-3、Caspase-9活化。
Aim: To study the effects of inhibiting Akt signaling pathway on the growth, cell cycle and apoptosis of fibroblasts in pathological scar.Methods: Human pathological scar fibroblasts were isolated and cultured in vitro. After treatment with different concentrations of Akt signaling pathway inhibitor LY294002, cell proliferation was detected by MTT, and IC50 was calculated. After treatment by IC50 concentration of LY294002, cell cycle and apoptosis were detected by flow cytometry, and the levels of Cleaved Caspase-3, Cleaved Caspase-9, Akt, and p-Akt were detected by Western blot.Results: The viability decreased significantly after LY294002 treatment with different concentrations(P<0.001), and the IC50 was 30 mg/L. After treatment by 30 mg/L LY294002, compared with 0 mg/L group, the G0/G1 phase cells were significantly increased(P<0.001), the apoptosis rate was elevated, the levels of Cleaved Caspase-3 and Cleaved Caspase-9 were significantly higher, while the level of p-Akt was significantly lower(P<0.001).Conclusion: Inhibition Akt signaling pathway could inhibit fibroblasts growth in pathological scar, block cell cycle, promote cell apoptosis, and promote the activation of Caspase-3 and Caspase-9