人核心蛋白聚糖真核表达载体的构建及其体内外抗瘤效应研究

摘要

Objective To construct the eukaryotic expression vector pcDNA3.1(+) - DCN and to explore its effects on sarcoma cell line S180 cells in vitro and in vivo. Methods After cDNA sequence of DCN was obtained from the pPIC9K-DCN with PCR,the eukaryotic expression vector pcDNA3.1(+) - DCN was constructed by subcloning DCN cDNA into pcDNA3.1(+) and then identified by double enzyme digestion,PCR and DNA sequencing.Recombinant plasmid pcDNA3.1(+) - DCN was transfected into S180 cells by liposome transfection,with transfected empty plasmid pcDNA3.1(+) and untransfected cells as control groups.Different concentrations of G418 were used to screen the positive transformants.RT-PCR was performed to determine hDCN mRNA expression in transfected S180 cells.Cell cycle and apoptosis of transfected S180 cells were analyzed by flow cytometry(FCM).The effects of DCN on tumor growth were observed after inoculated with pcDNA3.1(+)- DCN/S180,pcDNA3.1(+)/S180 and S180 cells in mice,respectively.The pathological evaluation of tumor tissues was performed after all mice were sacrificed. Results The eukaryotic expression vector of human decorin (hDCN) gene was constructed successfully.Apoptosis indices of S180 cells transfected with DCN significantly increased,with the higher proportion of cells in G0/G1 phase and the lower proportion in G2/M and S phase compared to the controls.The cell growth of tumor was inhibited in mice injected with pcDNA3.1(+)- DCN/S180 compared with that of other controls,and the tumor volume also reduced. Conclusion The recombinant eukaryotic expression vector pcDNA3.1(+)- DCN is constructed successfully.This recombinant eukaryotic expression vector can induce sarcoma cells apoptosis in vitro,which is associated with G0/G1 arrest,and exert the inhibitory effect on sarcoma in vivo.%目的 构建人核心蛋白聚糖(human decorin,hDCN)pcDNA3.1(+)真核表达载体,并探讨其对S180细胞在体内、体外的抗肿瘤效应及其作用机制.方法 用PCR法从pPIC9K-DCN扩增hDCN核心蛋白分子编码序列的全长cDNA,与pcDNA3.1(+)载体连接.将重组pcDNA3.1(+)- DCN质粒通过脂质体转染法转入小鼠肿瘤细胞S180中,用G418筛选出阳性克隆细胞,RT-PCR、双酶切及测序鉴定.流式细胞仪对转染了重组质粒pcDNA3.1(+) - DCN、空质粒pcDNA3.1(+)的S180细胞和未转染的S180细胞进行细胞凋亡检测和细胞周期分析.分别注射等量的pcDNA3.1(+) - DCN/S180、pcDNA3.1(+)/S180的细胞和未转染质粒的S180细胞到随机分组的小鼠腋部皮下,观察三组小鼠的肿瘤生长情况;处死三组小鼠后,分别剥离肿瘤组织做病理学检查.结果 转染了DCN的S180细胞凋亡指数明显增高,G0/G1期细胞百分率明显高于其他两组,G2/M期、S期细胞百分率分别明显低于其他两组(P<0.01).与注射pcDNA3.1(+)/S180和S180的小鼠相比,注射pcDNA3.1(+) - DCN/S180的小鼠肿瘤生长速度明显减慢,肿瘤体积小于其他两组(P<0.01).结论 成功构建了人核心蛋白聚糖真核表达载体.体外实验结果 显示转染该表达载体的S180细胞凋亡指数明显增高,且凋亡多发生在G0/G1期;转染DCN表达载体的S180细胞在实验动物体内成瘤性降低,提示转染DCN在实验动物体内外都能表现抑瘤效应.