康莱特注射液对吉非替尼诱导人肺腺癌A549细胞株凋亡影响的实验研究

目的 探讨康莱特注射液联合吉非替尼对人肺腺癌A549细胞株增殖和凋亡的影响。 方法 应用MTT法计算康莱特注射液作用于人肺腺癌A549细胞株48 h的半数抑制浓度（IC50），检测康莱特注射液（5、10、20、40、80、100、160 μl／ml）、吉非替尼（0.1、0.5、1.0、5、10、20、40 μmol／L）以及康莱特注射液（80 μl／ml）联合吉非替尼（0.1、0.5、1、5、10、20、40 μmol／L）作用于人肺腺癌A549细胞株24、48、72 h的增殖抑制率；流式细胞仪Annexin V/PI 双标法检测康莱特注射液、吉非替尼及两药联合作用于A549细胞48 h后的凋亡率；免疫细胞化学法检测各药物处理组作用于A549细胞48 h后FAS、AKT2蛋白表达；RT-PCR技术检测各组FAS mRNA、AKT2 mRNA的表达水平。均设未加药的A549细胞为空白对照组。结果 康莱特注射液作用于人肺腺癌A549细胞株48 h的IC50为80 μl／ml。康莱特注射液联合吉非替尼作用于A549细胞的增殖抑制率高于两单药组和空白对照组，且呈时间和浓度依赖性（P＜0.05）。两者的联合作用在一定浓度范围内呈现出协同的抗瘤效果。流式细胞术检测显示，两药联合组的细胞凋亡率显著高于两单药组和空白对照组（P＜0.05）。免疫细胞化学和RT-PCR检测显示，与空白对照组比较，康莱特组和两药联合组FAS蛋白和mRNA的表达水平均升高（P＜0.05），AKT2蛋白和mRNA表达水平均下调（P＜0.05）。结论 康莱特注射液联合吉非替尼对人肺腺癌A549细胞株有明显的促凋亡作用，康莱特注射液可能增加人肺腺癌A549细胞对于吉非替尼的敏感性。

Effects of kanglaite injection on the apoptosis induced by gefitinib in lung adenocarcinoma A549 cell line

Objective To investigate the effects of Kanglaite injection（KLT）combined with gefitinib on the lung adenocarcinoma A549 cell proliferation and apoptosis in vitro. Methods MTT assay was applied to calculate the half inhibitory concerntration（IC50） of KLT acting on human lung adenocarcinoma A549 cells for 48 h，and detect the inhibition rates by treating A549 cells with KLT（5，10，20，40，80，100，160 μl／ml），gefitinib（0.1，0.5，1.0，5，10，20，40 μmol／L） and KLT（80 μl／ml） combined with different doses of gefitinib for 24，48，72 h. Flow cytometry（FCM） was employed to detect the cell apoptosis for 48 h. Immunocytochemical and RT-PCR methods were used to detect the protien and mRNA express of AKT2 and FAS，respectively. Cells without drug was set as blank control group. ResultsMTT assay showed that IC50 of KLT acting on A549 cells for 48 h was 80 μl／ml. Compared with both single drug groups and control group， KLT combined with gefitinib could significantly inhibit the proliferation of A549 cells in a time-and dose-dependent manner. FCM showed that the cell apoptotic rates of combined group higher than those of both single drug groups and control group（P＜0.05）. Immunocytochemical and RT-PCR detection showed that compared with the control group， the FAS protein and mRNA levels of the KLT in combined group were increased， but AKT2 decreased（P＜0.05）. Conclusion The combination of KLT and gefitinib can induce cell apoptosis significantly. KLT may increase the sensitivity of the human lung adenocarcinoma cell line A549 for gefitinib.