CAJ | 학술논문

目的：构建P53凋亡刺激蛋白（ASPP）家族的抑制成员iASPP的真核表达载体，并将其通过脂质体转染到结肠癌细胞株SW480及Lovo中，观察转染前后iASPP 表达变化及其对细胞凋亡的影响。方法将解放军第十六医院检验科经测序鉴定的pMD19-T-iASPP质粒亚克隆至真核表达质粒pcDNA3．1（＋），构建重组真核表达质粒pcDNA3．1（＋）-iASPP ，测序鉴定后用脂质体将重组质粒转染至结肠癌细胞株SW480及Lovo中，用逆转录聚合酶链反应（RT-PCR）检测iASPP的表达以及用流式细胞仪检测细胞凋亡的变化情况。结果重组表达质粒pcDNA3．1（＋）-iASPP ，经酶切测序与GenBank上记录的人iASPP cDNA序列（gi 60457962）完全一致。经pcDNA3．1（＋）-iASPP质粒转染的结肠癌细胞株SW480及Lovo的iASPP mRNA表达增高，细胞凋亡率下降。结论成功构建了重组表达质粒pcDNA3．1（＋）-iASPP ，并成功在结肠癌细胞株SW480及Lovo中获得了表达，细胞株的凋亡率下降，提示抑制iASPP高表达有可能成为恢复P53抑癌功能的新策略。
목적：구건P53조망자격단백（ASPP）가족적억제성원iASPP적진핵표체재체，병장기통과지질체전염도결장암세포주SW480급Lovo중，관찰전염전후iASPP 표체변화급기대세포조망적영향。방법장해방군제십륙의원검험과경측서감정적pMD19-T-iASPP질립아극륭지진핵표체질립pcDNA3．1（＋），구건중조진핵표체질립pcDNA3．1（＋）-iASPP ，측서감정후용지질체장중조질립전염지결장암세포주SW480급Lovo중，용역전록취합매련반응（RT-PCR）검측iASPP적표체이급용류식세포의검측세포조망적변화정황。결과중조표체질립pcDNA3．1（＋）-iASPP ，경매절측서여GenBank상기록적인iASPP cDNA서렬（gi 60457962）완전일치。경pcDNA3．1（＋）-iASPP질립전염적결장암세포주SW480급Lovo적iASPP mRNA표체증고，세포조망솔하강。결론성공구건료중조표체질립pcDNA3．1（＋）-iASPP ，병성공재결장암세포주SW480급Lovo중획득료표체，세포주적조망솔하강，제시억제iASPP고표체유가능성위회복P53억암공능적신책략。
Objective Construct the eukaryotic expression vector of inhibitory member of the ASPP family (iASPP) and trans-fect it into colon carcinoma cell lines SW480 and Lovo by liposome .Then observe the expression of iASPP and detect the cell apop-tosis by flow cytometry .Methods The amplified PCR product was digested and inserted into pMD19-T simple vector and sub-cloned into eukaryotic expression vector pcDNA3 .1(+ ) .The recombinant eukaryotic expression plasmid pcDNA3 .1(+ )-iASPP was transfected into colon carcinoma cell lines SW480 and Lovo by liposome ,the iASPP expression was analyzed by RT-PCR .The cell apoptosis was detected by FCM .Results The eukaryotic expression plasmid pcDNA3 .1(+ )-iASPP was constructed success-fully ,the gene squence of iASPP was consistent with that reported (gi 60457962) in GenBank .The mRNA expression levels of iASPP gene of SW480 and Lovo cell lines which transfect the positive plasmid were increased ,and the cell apoptosis rates were de-creased .Conclusion We successfully constructed the recombinant expression plasmid pcDNA 3 .1(+ )-iASPP ,and the plasmid were successfully expressed in colon carcinoma cell lines SW 480 and Lovo ,the cell apoptosis rates of those cell lines were decreased .These facts indicated that reducing the high expression of iASPP may be a new strategy to renew the abilities of P 53 tumor suppressor .