CAJ | 학술논문

目的观察羧胺三唑对肺腺癌细胞A549细胞自噬的影响，探究自噬调节在羧胺三唑抗肿瘤增殖作用中的角色。方法 SRB法测定羧胺三唑对A549细胞的体外增殖抑制作用，Logit软件计算半数抑制浓度（ IC50）；AnnexinV-FITC/PI双染流式细胞术测定羧胺三唑给药48 h诱导A549细胞的凋亡率；Western blot检测羧胺三唑给药24 h后LC3-Ⅱ蛋白水平；EGFP-LC3质粒转染观察细胞自噬泡斑点情况；透射电子显微镜（ TEM）实验观察细胞自噬超微结构；采用溶酶体蛋白酶抑制剂评价羧胺三唑对自噬潮的影响；激光共聚焦显微镜结合MDC荧光染料观察细胞自噬溶酶体水平；流式细胞术结合MDC染色检测羧胺三唑对细胞自噬溶酶体的增加百分率。结果羧胺三唑剂量依赖性地和时间依赖性地抑制A549细胞增殖，72 h的IC50＝（13.81±1.15）μmol/L，83 h的IC50＝（5.86±1.06）μmol/L。羧胺三唑40μmol/L诱导A549细胞凋亡率为（18.7±1.9）％，P＜0.01。羧胺三唑能增加A549细胞LC3-Ⅱ蛋白表达水平，增加细胞EGFP＋-LC3亮点数目。电镜超微结构结果表明羧胺三唑作用24 h引起A549细胞自噬泡增多。在溶酶体蛋白酶抑制剂E64 d/pepstatinA存在下，羧胺三唑作用24 h未进一步升高LC3-Ⅱ蛋白水平。羧胺三唑作用A549细胞24 h显著增强自噬溶酶体水平（ P＜0.05）。结论羧胺三唑能够抑制A549细胞增殖，其作用机制可能与抑制A549细胞自噬体的降解有关。
목적관찰최알삼서대폐선암세포A549세포자서적영향，탐구자서조절재최알삼서항종류증식작용중적각색。방법 SRB법측정최알삼서대A549세포적체외증식억제작용，Logit연건계산반수억제농도（ IC50）；AnnexinV-FITC/PI쌍염류식세포술측정최알삼서급약48 h유도A549세포적조망솔；Western blot검측최알삼서급약24 h후LC3-Ⅱ단백수평；EGFP-LC3질립전염관찰세포자서포반점정황；투사전자현미경（ TEM）실험관찰세포자서초미결구；채용용매체단백매억제제평개최알삼서대자서조적영향；격광공취초현미경결합MDC형광염료관찰세포자서용매체수평；류식세포술결합MDC염색검측최알삼서대세포자서용매체적증가백분솔。결과최알삼서제량의뢰성지화시간의뢰성지억제A549세포증식，72 h적IC50＝（13.81±1.15）μmol/L，83 h적IC50＝（5.86±1.06）μmol/L。최알삼서40μmol/L유도A549세포조망솔위（18.7±1.9）％，P＜0.01。최알삼서능증가A549세포LC3-Ⅱ단백표체수평，증가세포EGFP＋-LC3량점수목。전경초미결구결과표명최알삼서작용24 h인기A549세포자서포증다。재용매체단백매억제제E64 d/pepstatinA존재하，최알삼서작용24 h미진일보승고LC3-Ⅱ단백수평。최알삼서작용A549세포24 h현저증강자서용매체수평（ P＜0.05）。결론최알삼서능구억제A549세포증식，기작용궤제가능여억제A549세포자서체적강해유관。
Objective To explore the influence of carboxyamidotrizazole ( CAI ) on autophagy in lung adenocarcinoma A549 cells.Methods The proliferation inhibition rate was tested by SulforhodamineB (SRB)method in A549 cells,calculating IC50 value by Logit.Apoptosis rates were tested by AnnexinV-FITC/PI double staining with flow cytometry in A549 cells treated with CAI for 48 h.Autophagy activity was detected using analysis of LC 3-Ⅱprotein expression by western blot method ,EGFP+-LC3 puncta changes by EGFP-LC3 plasmid transfection and laser confocal fluorescence microscopy .We also used transmission electron microscopy to observe autophagic vacuoles at ultrastructure level.Additionally,we monitored authphagic influx by lysosomal inhibitor pepstaitinA and E 64d to distinguish whether autophagosome accumulation was due to autophagy induction or a blockage in downstream steps . The change of autolysosomes in quantity was tested by MDC staining with Laser confocal microscopy and flow cytometry method .Results CAI suppressed the proliferation of A 549 cells in a time-and dose-dependent manner . IC50 value was(13.81 ±1.15)μmol/L for 72 h and(5.86 ±1.06)μmol/L for 83 h.Apoptosis rate induced by CAI40 μmol/L in A549 for 48 h was(18.7 ±1.9)%,P<0.01.Autophagy protein marker LC3-Ⅱand EGFP +-LC3 puncta number increased after treatment with CAI for 24 h.Autophagic vacuoles(AVs)also increased revealed in the TEM observation .Under the presence of pepstatinA and E 64 d the addition of CAI for 24 h didn′t increased LC3-Ⅱprotein expression .CAI significantly increased MDC mean fluorescence intensity which means an enhancement of the amount of autolysosomes in A549 cells(P<0.05).Conclusion CAI inhibits the proliferation of A549 cells may be caused by the blockage of accumulated autolysosomes degradation .