巴豆生物碱对肺腺癌细胞凋亡的影响及机制研究

Influence and mechanism study of crotonic alkaloids on apoptosis of lung adenocarcinoma cell

目的研究巴豆生物碱(CA)对肺腺癌细胞(A549)增殖和凋亡的影响,推测其抗肺癌作用机制。方法体外培养肺腺癌细胞(A549)。用终浓度分别为0(对照组)、10、50、100μg/mL的CA组处理A549细胞24 h后,于相差显微镜下观察细胞的一般形态结构、脱落情况等;MTT法检测细胞的生长活性;流式细胞仪检测细胞凋亡率;酶联免疫法(ELISA)和RT-PCR法检测凋亡相关蛋白及基因Bcl-2、Bax的表达。结果 10、50、100μg/mL的CA均能抑制A549细胞增殖,其抑制率与作用剂量相关。流式细胞术检测显示不同剂量CA作用于A549细胞24 h后,100μg/mL的CA可引起细胞凋亡,其余剂量未见凋亡;100μg/mL的CA组细胞凋亡率为(45.940±0.031)%,对照组细胞凋亡率为(3.450±0.012)%,差异有统计学意义(P<0.05)。ELISA提示不同剂量CA作用于A549细胞24 h后,100μg/mL的CA组促凋亡蛋白Bax为8.940±0.211,较对照组(4.450±0.131)表达增加;抑凋亡蛋白Bcl-2为4.860±0.132,较对照组(7.390±0.181)表达减少,差异有统计学意义(P<0.05)。RT-PCR显示100μg/mL的CA组Bax mRNA为6.881±0.611,较对照组(3.417±0.461)表达增加;Bcl-2 mRNA为2.812±0.342,较对照组(4.243±0.731)表达减少,差异有统计学意义(P<0.05)。结论 CA能抑制A549细胞的生长并促进其凋亡,其发生与Bax蛋白及基因的表达上调和Bcl-2蛋白及基因的表达下调有关。CA抗肺癌的作用机制可能通过Bax/Bcl-2途径促进肿瘤细胞凋亡,从而为临床治疗肺癌提供新的思路和途径。

Objective To confer the mechanism of action through researching the effect of the croton alkaloids（CA） on proliferation and apoptosis of lung adenocarcinoma cells（A549）.Methods The A549 cells were exposed into CA at various doses（0,10,50,100 μg/mL） for 24 h.The morphology and growth feature of the A549 cells were observed by the phase contrast microscope.The cell growth activity was detected through the MTT method.The cell apoptosis rate was detected by the flow cytometry instrument.The expression of gene Bcl-2 and Bax were detected through the ELISA and RT-PCR methods.Results The doses of 10,50,100 μg/mL could inhibit the A549 cells proliferation,the inhibition ratio was correlated with concentration.Different doses CA on A549 cells after 24 h,the result of flow cytometry test showed that only CA group（100 μg/mL） could induce cell apoptosis,the other dose was not apoptosis.The apoptosis rate of CA group（100 μg/mL） was（45.940±0.031）%,the apoptosis rate of control group was（3.450±0.012）%,the difference between two groups was significant（P〈0.05）.Different doses of CA on A549 cells after 24 h,the result of ELISA showed that pro-apoptotic protein Bax of CA group（100 μg/mL） was 8.940±0.211,express increased than that of control group（4.450±0.131）;suppression of apoptosis protein Bcl-2 of CA group（100 μg/mL） was 4.860±0.132,express decreased than that of control group（7.390±0.181）,the differences between two groups were significant（P〈0.05）.RT-PCR showed that Bax mRNA of CA group（100 μg/mL） was 6.881±0.611,express increased than that of control group（3.417±0.461）;Bcl-2 mRNA of CA group（100 μg/mL） was 2.812±0.342,express decreased than that of control group（4.243±0.731）,the differences between two groups were significant（P〈0.05）.Conclusion CA can inhibit the A549 cells growth and promote its apoptosis which through up-regulated the Bax gene and down-regulated the Bcl-2 gene.CA may through regulate the Bax/Bcl-2gene induced apoptosis,which may provide new approaches for clinical treatment of lung cancer.