CAJ | 학술논문

目的探讨抑制乙酰肝素酶表达对人肺腺癌细胞A549增殖、侵袭和凋亡的影响.方法构建靶向人乙酰肝素酶基因短发夹状双链RNA(shRNA)真核表达质粒(pshRNA-Hpa),用脂质体将其转入A549细胞,建立稳定转染细胞株系,经逆转录.PCR和Western blot法检测转染效率后,分别用四甲基偶氮唑蓝(MTT)、Transwell侵袭实验和流式细胞术(AnnexinV-FITC/PI双染法)检测其对A549细胞增殖、侵袭和凋亡的影响.采用Western blot法检测转染后磷酸化蛋白激酶B(PKB/Akt-Ser473)的表达.结果转染pshRNA-Hpa的A549细胞乙酰肝素酶mRNA及蛋白表达明显低于未处理A549细胞,抑制率分别为54.09%和48.26%,细胞增殖速度减缓,侵袭能力降低,流式细胞术显示早期细胞凋亡率达(12.53±0.34)%,较空白对照组、空载体转染细胞组和阴性对照组明显增加(P＜0.01).同阴性对照质粒相比,转染pshRNA-Hpa的A549细胞Akt-Ser473的磷酸化水平降低52.15%.结论靶向乙酰肝素酶重组shRNA质粒pshRNA-Hpa能有效抑制A549细胞乙酰肝素酶的表达,并可能通过下调磷酸化蛋白激酶B途径抑制细胞的增殖侵袭、促进细胞的凋亡.
목적 탐토억제을선간소매표체대인폐선암세포A549증식、침습화조망적영향.방법 구건파향인을선간소매기인단발협상쌍련RNA(shRNA)진핵표체질립(pshRNA-Hpa),용지질체장기전입A549세포,건립은정전염세포주계,경역전록.PCR화Western blot법검측전염효솔후,분별용사갑기우담서람(MTT)、Transwell침습실험화류식세포술(AnnexinV-FITC/PI쌍염법)검측기대A549세포증식、침습화조망적영향.채용Western blot법검측전염후린산화단백격매B(PKB/Akt-Ser473)적표체.결과 전염pshRNA-Hpa적A549세포을선간소매mRNA급단백표체명현저우미처리A549세포,억제솔분별위54.09%화48.26%,세포증식속도감완,침습능력강저,류식세포술현시조기세포조망솔체(12.53±0.34)%,교공백대조조、공재체전염세포조화음성대조조명현증가(P＜0.01).동음성대조질립상비,전염pshRNA-Hpa적A549세포Akt-Ser473적린산화수평강저52.15%.결론 파향을선간소매중조shRNA질립pshRNA-Hpa능유효억제A549세포을선간소매적표체,병가능통과하조린산화단백격매B도경억제세포적증식침습、촉진세포적조망.
objective To investigate the effects of heparanase expression inhibition on the proliferation,invasiveness and apoptosis of human lung adenocarcinoma cell line A549 ceHs.nethods Recombinant eukaryotic expression plasmid pshRNA-Hpa targeting human heparanase gene was constructed.A549 cells were cultured in DMEM and transfected with pshRNA-Hpa.The expression of heparanase mRNA and protein were examined bv RT-PCR and Western blot.The proliferation.invasiveness and apoptotic rates of A549 cells were determined bv MTT method.matrigel invasion assays and flow cytometry respectively.Results The expression levels of heparanase mRNA and protein were down-regulated in A549 transfected with pshRNA-Hpa.The number of cells penetrating matrigel and the proliferation ability of A549 cells transfected with pshRNA-Hpa were reduced significantly compared to the control cells.The apoptotic rate of A549 cells transfected with pshRNA-Hpa was 12.53%±0.34%.being siguificanfly higher tIlan that of the control cells(both P＜0.01).Western-blot showed that inhibition of heparanase expression led to reduced Akt phosphorylation.Conclusions The recombinant plasmid pshRNA-Hpa effectively inhibited the expression of heparanase,thus suppressing the proliferation and invasion and inducing apoptosis of A549 cells.The effects may be due to the down-regulation of Akt phosphorylafion level.