重组人血管内皮抑制素注射液抑制非小细胞肺癌细胞A549增殖及诱导凋亡研究

Effect of endostar on proliferation and apoptosis of non-small lung cancer cell of A549

目的探讨恩度(重组人血管内皮抑制素注射液)对非小细胞肺癌A549细胞的增殖抑制及诱导凋亡作用机制。方法培养肿瘤细胞,分别加入浓度为25、50、100μg/mL的恩度,对照组则加入等量的培养液,运用MTT法检测A549细胞细胞活力;采用瑞氏-吉姆萨染色法检测A549细胞形态学变化;使用流式细胞仪在PI染色条件下实施细胞周期分析与凋亡判定;运用Western blot法检测Livin与Caspase-9蛋白的表达。结果恩度通过时间与剂量依赖方式来抑制A549细胞的增殖,在48、72、96h的时间点上抑制A549细胞增殖50%的药物浓度(IC50)分别为(54.23±5.47)、(14.86±3.15)及(6.37±3.83)μg/mL;恩度可以诱导肺癌细胞凋亡,形态学表现为产生凋亡小体和流式细胞仪所检测到的亚二倍体凋亡峰,细胞凋亡率48h分别为(2.87±0.71)%、(4.92±1.32)%及(7.41±1.76)%;72h分别为(4.73±0.84)%、(9.83±1.75)%及(18.12±1.62)%,与对照组比较差异均有统计学意义(P均<0.05)。Western blot检测结果显示,浓度为25、50、100μg/mL的恩度分别作用A549细胞48h,Livin蛋白表达分别下调至作用前的(62.35±8.12)%、(28.73±5.45)%及(21.84±5.84)%,与对照组比较差异均有统计学意义(P均<0.05);25μg/mL的恩度作用A549细胞48h仅检测到非活化状态的Pro-Caspase-9,而浓度为50、100μg/mL的恩度可检测到Caspase-9的活化亚基,且随浓度增大活化程度愈明显。结论恩度可抑制肺癌A549细胞的增殖,并诱导A549细胞凋亡。恩度可能通过抑制Livin的表达及活化Caspase-9发挥肿瘤细胞生长抑制作用。

Objective To investigate the mechanism of inhibition and apoptosis induction of Endostar （re -combinant human endostatin injection） on proliferation of non-small lung cancer cell A549. Methods Cul-tured tumor cells were added with 25 , 50, 100 μg/mL Endostar, A549 cell activity were detected by MTT method; A549 cell morphological changes were observed by Wright Giemsa staining method; the analysis and apoptosis determination of cell cycle in PI dyeing condition were implemented by flow cytometry; the expression of Livin and Caspase-9 protein were detected by Western blot method. Results Endostar inhibi-ted the proliferation of A549 cells through time and dose-dependent manner, the IC50 of Endostar on the proliferation of A549 at 48 h, 72 h and 96 h were respectively （54.23± 5.47） , （14.86 ± 3.15） and （6.37 ± 3. 83） jug/mL; Endostar could induce apoptosis of lung cancer cells by observing apoptotic body morphologi-cally and subdiploid apoptotic peak with flow cytometry, the apoptosis rate of 25, 50 and 100 jug/mL En- dostar at 48 h were （2.87±0.71）%, （4.92±1.32）% and （7.41±1.76）% respectively; at 72 h were （4.73 ±0.84）% , （9.83 ± 1.75）% and （18.12±1.62） % respectively, which were significant differences to that of the control group （P 〈0.05）. The 3 concentrations of Endostar down-regulated Livin protein expression of A549 cells at 48 h by （62.35 ± 8.12）%, （28.73 ±5.45） % and （21.84±5.84） % of pre-administration re-spectively, which were significant differences to that of the control group （P〈0.05）. Non activated state of Pro-Caspase-9 could be detected by 25 μg/mL Endostar at 48 h, while activated state of Caspase-9 sub-units could be detected at 50 and 100 μg/mL Endostar at 48 h, which degrees of activation was in dose-de-pendent manner. Conclusion Endostar inhibits the proliferation of lung cancer A549 cells, perhaps through inhibition of the Livin expression and activation of Caspase-9.