壳寡糖抑制肺癌细胞系恶性增殖相关机制的探讨

Mechanism of Chitooligosaccharide Inhibiting Malignant Proliferation of Lung Cancer Cell Line Based on p53 Mediated Autophagy Pathway

目的探讨壳寡糖抑制肺癌细胞系恶性增殖的相关机制。方法选择肺癌细胞系A549随机分为阴性对照组、壳寡糖低浓度组、壳寡糖中浓度组、壳寡糖高浓度组,并分别予以0、1、2、5 mg·ml^(-1)壳寡糖处理。观察于24 h、48 h、72 h时各组A549细胞增殖抑制率,比较各组A549细胞凋亡率、凋亡相关蛋白[Bcl-2、Bax、生存素(Survivin)]及p53(p53、p-p53)通路相关蛋白表达水平。结果在24 h、48 h、72 h时,壳寡糖浓度组A549细胞系增殖抑制率较阴性对照组显著升高,且呈时间浓度依赖性(P<0.05)。壳寡糖浓度组A549细胞凋亡较阴性对照组显著升高,且呈浓度依赖性(P<0.05)。阴性对照组A549细胞染色较淡,表现为暗蓝色;壳寡糖浓度组A549细胞发生凋亡,出现核固缩、变小、核碎裂等现象,且和染色较深,表现为亮蓝色,同胞内及胞周存在颗粒状深染物质。壳寡糖浓度组A549细胞Bcl-2、Survivin蛋白表达水平均较阴性对照组显著降低,Bax蛋白表达水平均较阴性对照组显著升高,且呈浓度依赖性(P<0.05)。壳寡糖浓度组A549细胞中p53、p-p53、beclin 1蛋白表达水平阴性对照组显著升高,且呈浓度依赖性(P<0.05)。结论壳寡糖可抑制肺癌细胞系恶性增殖并促进细胞凋亡,其作用机制可能是通过调节p53介导的自噬通路进而上调p53、p-p53、beclin 1蛋白表达水平实现的。

Objective To explore the mechanism of chitooligosaccharide inhibiting malignant proliferation of lung cancer cell line based on p53 mediated autophagy pathway.Methods Lung cancer cell line A549 was randomly divided into negative control group,low concentration chitooligosaccharide group,medium concentration chitooligosaccharide group and high concentration chitooligosaccharide group,and treated with 0,1,2 and 5 mg·ml^(-1) chitooligosaccharide respectively.The proliferation inhibition rate of A549 cells was observed at 24h,48h and 72h,and the apoptosis rate,the expression levels of apoptosis related proteins[Bcl-2,Bax,survivin]and p53(p53,p-p53)pathway related proteins were compared.Results At 24h,48h and 72h,the proliferation inhibition rate of A549 cell line in chitooligosaccharide concentration group was significantly higher than that in negative control group(P<0.05).The apoptosis of A549 cells in chitosan oligosaccharide concentration group was significantly higher than that in negative control group(P<0.05).In the negative control group,the staining of A549 cells was light,showing dark blue;in the oligochitosan concentration group,A549 cells were apoptotic,with pyknosis,diminution,nuclear fragmentation,and deep staining,showing bright blue,with granular hyperchromatic substances in and around the siblings.Compared with the negative control group,the expression levels of Bcl-2 and survivin protein in the chitosan oligosaccharide concentration group were significantly decreased,and the expression levels of Bax protein in the chitosan oligosaccharide concentration group were significantly increased in a concentration dependent manner(P<0.05).The expression levels of p53,p-p53 and Beclin 1 protein in A549 cells in chitosan oligosaccharide concentration group were significantly higher than those in negative control group(P<0.05).Conclusion Chitooligosaccharide can inhibit the malignant proliferation and promote apoptosis of lung cancer cell line,and its mechanism may be through regulating the autophagy pathway mediated by p53 and then up regulating the expression levels of p53,p-p53 and Beclin 1.