上调转录因子Sp1对胃癌细胞MKN-45增殖的影响

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.

Effects of up-regulation of transcription factor Sp1 on proliferation of MKN-45 gastric cancer cells

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.