中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2011年
11期
1903-1905
,共3页
周尘飞%计骏%蔡劬%于颖彦%陈雪华%刘炳亚%张俊%朱正纲
週塵飛%計駿%蔡劬%于穎彥%陳雪華%劉炳亞%張俊%硃正綱
주진비%계준%채구%우영언%진설화%류병아%장준%주정강
胃癌%Sp1%真核表达载体%细胞增殖
胃癌%Sp1%真覈錶達載體%細胞增殖
위암%Sp1%진핵표체재체%세포증식
Gastric cancer%Sp1%Eukaryotic expression vector%Cell proliferation
目的 构建pEGFP-Sp1真核表达载体,转染Sp1低表达胃癌细胞株MKN-45,观察转染后细胞内Sp1表达及其体外增殖能力.方法 PCR扩增Sp1 (2337 bp)全长序列,连接至pEGFP-N1质粒(4700 bp),测序鉴定无误后,以Lipofectamine 2000转染胃癌细胞株MKN-45.细胞免疫组织化学染色定位Sp1表达部位,PCR扩增及Western blot检测细胞Sp1表达.CCK-8法检测细胞体外增殖能力.结果构建pEGFP-Sp1表达质粒并转染胃癌细胞株,外源性Sp1定位于MKN-45细胞核中并稳定表达.体外培养3d后,MKN-45-Sp1增殖能力明显高于MKN-45-N1及MKN-45细胞(MKN-45-Sp1比MKN-45-N1及MKN-45,P＜0.05).结论 构建pEGFP-Sp1真核表达载体,稳定上调胃癌细胞株MKN-45中Sp1表达,转染后胃癌细胞株体外增殖能力明显增高.
目的 構建pEGFP-Sp1真覈錶達載體,轉染Sp1低錶達胃癌細胞株MKN-45,觀察轉染後細胞內Sp1錶達及其體外增殖能力.方法 PCR擴增Sp1 (2337 bp)全長序列,連接至pEGFP-N1質粒(4700 bp),測序鑒定無誤後,以Lipofectamine 2000轉染胃癌細胞株MKN-45.細胞免疫組織化學染色定位Sp1錶達部位,PCR擴增及Western blot檢測細胞Sp1錶達.CCK-8法檢測細胞體外增殖能力.結果構建pEGFP-Sp1錶達質粒併轉染胃癌細胞株,外源性Sp1定位于MKN-45細胞覈中併穩定錶達.體外培養3d後,MKN-45-Sp1增殖能力明顯高于MKN-45-N1及MKN-45細胞(MKN-45-Sp1比MKN-45-N1及MKN-45,P＜0.05).結論 構建pEGFP-Sp1真覈錶達載體,穩定上調胃癌細胞株MKN-45中Sp1錶達,轉染後胃癌細胞株體外增殖能力明顯增高.
목적 구건pEGFP-Sp1진핵표체재체,전염Sp1저표체위암세포주MKN-45,관찰전염후세포내Sp1표체급기체외증식능력.방법 PCR확증Sp1 (2337 bp)전장서렬,련접지pEGFP-N1질립(4700 bp),측서감정무오후,이Lipofectamine 2000전염위암세포주MKN-45.세포면역조직화학염색정위Sp1표체부위,PCR확증급Western blot검측세포Sp1표체.CCK-8법검측세포체외증식능력.결과구건pEGFP-Sp1표체질립병전염위암세포주,외원성Sp1정위우MKN-45세포핵중병은정표체.체외배양3d후,MKN-45-Sp1증식능력명현고우MKN-45-N1급MKN-45세포(MKN-45-Sp1비MKN-45-N1급MKN-45,P＜0.05).결론 구건pEGFP-Sp1진핵표체재체,은정상조위암세포주MKN-45중Sp1표체,전염후위암세포주체외증식능력명현증고.
Objective To construct the eukaryotic expression vector of pEGFP-Sp1,and transfect MKN-45 gastric cancer cell line with low expression of Sp1,observing proliferation in vitro after transfection.Methods The sequence of Sp1 (2337 bp) was obtained by polymerase chain reaction (PCR) amplification.The PCR product was then inserted into the pEGFP-N1 vector (4700 bp),constructing the pEGFP-Sp1 vector.After confirming the DNA sequence,the pEGFP-Sp1 was transfected by Lipofectamine 2000 into MKN-45 gastric cell line which was selected by RT-PCR and Western blotting.The expression and localization of pEGFP-Sp1 were observed by fluorescence microscope and immunohistochemistry.PCR and Western blotting were used to detect the mRNA and protein levels of Sp1 respectively.Proliferation of tumor cells was measured by using CCK8.Results The recombinant vector pEGFP-Sp1 was transfected and expressed in MKN-45 gastric cancer cells.Sp1 protein was located in the nuclei.The proliferation ability of MKN-45-Sp1,on the third day in vitro,was significantly higher than MKN-45-N1 and MKN-45 cells (MKN-45-Sp1 vs.MKN-45-N1 and MKN-45,P＜0.05).Conclusion The constructed pEGFP-Sp1 expression vector can up-regulate the Sp1 expression in gastric cancer cells,and enhance the proliferation ability of in vitro.
