过表达核心蛋白聚糖基因对胆管癌细胞周期及凋亡的影响  

Effect of decorin gene overexpression on cell cycle and apoptosis of QBC939 cells

作　　者：王伟林[1] 余翔[1] 李泉[1] 毛永欢 沈佳佳[1] 骆霞岗[1] 闻浩[1] 喻春钊[1] 

机构地区：[1]南京医科大学第二附属医院普外科,210011

出　　处：《中华实验外科杂志》2015年第1期69-71,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（30972910、81172269）;中国博士后一等基金资助项目（20060390294）;江苏省自然科学基金资助项目（BIC2011858）;江苏省六大人才高峰基金资助项目（WSW-032）

摘　　要：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.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

关 键 词：胆管癌 核心蛋白聚糖 细胞周期 脱噬作用 

分 类 号：R735.8[医药卫生—肿瘤]