CAJ | 학술논문

目的观察过表达核心蛋白聚糖(DCN)基因对胆管癌细胞周期及凋亡的影响.方法脂质体介导真核表达质粒pEGFP-DCN和空载体pEGFP-N1转入人胆管癌细胞株QBC939,Westernblot检测DCN蛋白的表达,实时定量反转录-聚合酶链反应(RT-qPCR)检测DCN mRNA的表达,克隆形成实验计算克隆形成率,噻唑蓝(MTT)法检测细胞增殖活力,流式细胞术检测细胞周期及凋亡.结果 RT-qPCR示过表达DCN相对空载体上调32.34倍;Western blot结果示过表达组DCN上调2.00倍.pEGFP-DCN转染组细胞克隆形成数[(210.9±19.3)个]显著低于空质粒转染组[(608.7±56.3)个],差异有统计学意义(P＜0.05).转染pEGFP-DCN细胞与空载体比较,细胞增殖能力显著降低,差异有统计学意义(P＜0.05).流式细胞周期检测显示,胆管癌细胞转染pEGFP-DCN后G1～ Go期细胞比例明显升高,而G2～S期细胞减少,细胞周期被阻滞在G1～Go期,凋亡分析转染pEGFP-DCN的QBC939细胞较转染空载体细胞凋亡显著增加,半胱氨酰天冬氨酸特异性蛋白酶(Caspase)-3明显增高,而B细胞淋巴瘤/白血病-2(bcl-2)的表达水平明显降低(0.787±0.068比1.276±0.157).结论转染DCN能够显著抑制胆管癌细胞的增殖,明显促进胆管癌细胞凋亡,而DCN促进胆管细胞凋亡与上调Caspase-3和下调bcl-2蛋白相关.
목적 관찰과표체핵심단백취당(DCN)기인대담관암세포주기급조망적영향.방법 지질체개도진핵표체질립pEGFP-DCN화공재체pEGFP-N1전입인담관암세포주QBC939,Westernblot검측DCN단백적표체,실시정량반전록-취합매련반응(RT-qPCR)검측DCN mRNA적표체,극륭형성실험계산극륭형성솔,새서람(MTT)법검측세포증식활력,류식세포술검측세포주기급조망.결과 RT-qPCR시과표체DCN상대공재체상조32.34배;Western blot결과시과표체조DCN상조2.00배.pEGFP-DCN전염조세포극륭형성수[(210.9±19.3)개]현저저우공질립전염조[(608.7±56.3)개],차이유통계학의의(P＜0.05).전염pEGFP-DCN세포여공재체비교,세포증식능력현저강저,차이유통계학의의(P＜0.05).류식세포주기검측현시,담관암세포전염pEGFP-DCN후G1～ Go기세포비례명현승고,이G2～S기세포감소,세포주기피조체재G1～Go기,조망분석전염pEGFP-DCN적QBC939세포교전염공재체세포조망현저증가,반광안선천동안산특이성단백매(Caspase)-3명현증고,이B세포림파류/백혈병-2(bcl-2)적표체수평명현강저(0.787±0.068비1.276±0.157).결론 전염DCN능구현저억제담관암세포적증식,명현촉진담관암세포조망,이DCN촉진담관세포조망여상조Caspase-3화하조bcl-2단백상관.
Objective To investigate the effects of decorin (DCN) gene overexpression on cell cycle and apoptosis of human cholangiocarcinoma cells.Methods The pEGFP-DCN plasmids and pEGFP-N1 control plasmids were transfected into the QBC939 cells using Lipofectamine 2000.The mRNA and protein expression of decorin in QBC939 cells transfected with plasmid overexpressing decorin was detected by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting respectively.The growth of QBC939 cells was detected by using colony-forming growth assays.Cell proliferation activity was detected by methyl thiazol tetrazolium (MTT).Cell cycle and apoptosis of cells were analyzed by flow cytometry (FCM).The expression of apoptosis-related protein,cysteinyl aspartate-specific protease-3 (Caspase-3) and B cell lymphoma/leukemia-2 (bcl-2) was detected by Western blotting.Results Compared to the control plasmids,RT-qPCR showed the mRNA expression of decorin in the pEGFP-DCN plasmids was increased by 32.34 fold.Western blotting showed the protein expression of decorin was increased by 2 fold.The growth of QBC939 cells was inhibited in cells transfected with pEGFP-DCN as compared to the empty vector.MTT assay showed a decrease of QBC939 cells proliferation in cells transfected with pEGFP-DCN as compared to the empty vector.Cell cycle analysis presented an obvious cell-cycle arrest at the G1-G0 phase and a decreased G2-S phase in cells transfected with pEGFP-DCN as compared to the negative control group.Flow cytometry detected an increase of the apoptotic cells in cells treated with pEGFP-DCN as compared to the empty vector.Western blotting identified that cleaved Caspase-3 was significantly increased,and bcl-2 was decreased in cells transfected with pEGFP-DCN (0.787 ± 0.068 vs.1.276 ± 0.157).Conclusion The overexpression of DCN gene could significantly promote the proliferation of cholangiocarinoma cells,which may be associated with the overexpression of Caspase-3 and downexpression of bcl-2.