清肺益气方对肺癌干细胞样细胞增殖的抑制作用及其机制研究

摘要

Objective It is to observe the inhibitory effect of Qingfei Yiqi Recipe on the proliferation of stem cell-like cells (CICs) in lung cancer and its possible mechanism. Methods Human lung cancer cell line A549 was cultured in vitro. The serum of Qingfei Yiqi Recipe containing medicine was used. The proliferation of lung cancer cells was detected by MTT colorimetric assay,cell apoptosis was detected by flow cytometry,the expression of the genes and proteins of white p53 and Caspase-3 were detected by real time fluorescence quantitative PCR and Western blot,the expression of VEGF and HIF-1α was detected by ELISA method,the invasion ability of lung cancer CICs in vitro was detected by Transwell chamber invasion test. Results The inhibitory rate of cell proliferation in the Qingfei Yiqi Recipe groups was significantly higher than that in the negative control group and the blank control group (P<0.05). With the increase of the concentration of serum containing drugs,its effect on inhibiting the proliferation of lung cancer cells gradually increased,with concentration-dependent manner,the IC50 of the Qingfei Yiqi Recipe was 80.12 μmol /mL,with the prolongation of the time of drug delivery, the morphology of lung cancer cells gradually shriveled and deformed,with necrosis and fall off. Compared with the blank control group and the negative control group,the apoptosis rate of the Qingfei Yiqi Recipe groups was significantly increased(P<0.05). The expression levels of mRNA and protein of Caspase-3 and p53 genes in the cells of Qingfei Yiqi Recipe group were significantly higher than those in the negative control group and the blank control group (all P<0.05). The expression level of VEGF and HIF-1α in the cell supernatant of Qingfei Yiqi Recipe group were significantly lower than those of the negative control group and the blank control group (all P<0.05). The number of invasive cells in the treatment group of Qingfei Yiqi Recipe was significantly lower than that of the negative control group and the blank control group(P<0.05). Conclusion Qingfei Yiqi Recipe has a significant inhibitory effect on CICs of lung cancer. Its mechanism may be play a role in anti-tumor proliferation mediated by multiple targets such as up-regulation the expression of apoptosis suppressor genes and protein p53 and Caspase-3,promote the apoptosis of lung cancer CICs and weaken the migration and invasiveness of CICs in lung cancer,inhibit the expression of VEGF and HIF-1α,and inhibit tumor angiogenesis.%目的 观察清肺益气方对肺癌干细胞样细胞(CICs)增殖的抑制作用及其可能的作用机制.方法 人肺癌细胞株A549经传代培养后,采用清肺益气方含药血清进行干预,采用MTT比色法检测肺癌细胞的增殖情况,流式细胞术检测细胞凋亡情况,实时荧光定量PCR法和Western blot法检测p53和Caspase-3基因和蛋白的表达,ELISA法检测 VEGF和HIF-1α的表达,Transwell小室侵袭实验法检测肺癌CICs体外侵袭能力变化.结果 清肺益气方组细胞增殖抑制率显著高于阴性对照组和空白对照组(P<0.05).且随着含药血清浓度的增加,其抑制肺癌细胞增殖的作用逐渐增强,呈浓度依赖性,根据公式计算得到清肺益气方的IC50为80.12 μmol/mL,且随着给药时间的延长,肺癌细胞形态逐渐皱缩变形,坏死脱落.与空白对照组及阴性对照组比较,清肺益气方组细胞凋亡率明显增加(P<0.05).清肺益气方处理组细胞中Caspase-3和p53基因mRNA和蛋白的表达水平均显著高于阴性对照组和空白对照组(P均<0.05).清肺益气方处理组细胞上清液中VEGF和HIF-1α的表达水平均显著低于阴性对照组和空白对照组(P均<0.05).清肺益气方处理组的侵袭细胞数显著低于阴性对照组和空白对照组(P均<0.05).结论 清肺益气方对肺癌CICs具有显著的抑制作用,其机制可能通过上调凋亡抑制基因和蛋白p53和Caspase-3的表达,促进肺癌 CICs的凋亡,削弱肺癌CICs的迁移与侵袭能力,并抑制VEGF和HIF-1α的表达,抑制肿瘤血管的生成等多个靶点发挥抗肿瘤增殖的作用.