CAJ | 학술논문

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.
목적 구건인핵심단백취당(human decorin,hDCN)pcDNA3.1(+)진핵표체재체,병탐토기대S180세포재체내、체외적항종류효응급기작용궤제.방법 용PCR법종pPIC9K-DCN확증hDCN핵심단백분자편마서렬적전장cDNA,여pcDNA3.1(+)재체련접.장중조pcDNA3.1(+)- DCN질립통과지질체전염법전입소서종류세포S180중,용G418사선출양성극륭세포,RT-PCR、쌍매절급측서감정.류식세포의대전염료중조질립pcDNA3.1(+) - DCN、공질립pcDNA3.1(+)적S180세포화미전염적S180세포진행세포조망검측화세포주기분석.분별주사등량적pcDNA3.1(+) - DCN/S180、pcDNA3.1(+)/S180적세포화미전염질립적S180세포도수궤분조적소서액부피하,관찰삼조소서적종류생장정황;처사삼조소서후,분별박리종류조직주병이학검사.결과 전염료DCN적S180세포조망지수명현증고,G0/G1기세포백분솔명현고우기타량조,G2/M기、S기세포백분솔분별명현저우기타량조(P<0.01).여주사pcDNA3.1(+)/S180화S180적소서상비,주사pcDNA3.1(+) - DCN/S180적소서종류생장속도명현감만,종류체적소우기타량조(P<0.01).결론 성공구건료인핵심단백취당진핵표체재체.체외실험결과 현시전염해표체재체적S180세포조망지수명현증고,차조망다발생재G0/G1기;전염DCN표체재체적S180세포재실험동물체내성류성강저,제시전염DCN재실험동물체내외도능표현억류효응.
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.