真核表达p27构建胃癌顺铂耐药细胞模型的研究

目的:构建p27真核表达载体并导入胃癌SGC-7901细胞获得稳定表达p27的稳定细胞株，以研究p27在胃癌SGC-7901细胞顺铂耐药中所发挥的功能。方法:以乳腺文库为模板，PCR扩增出p27编码区，并将其连接到pCDNA 3.0-Flag载体上，转染293T细胞后分别用定量PCR和Western blot检测其表达情况，并通过Western blot检测SGC-7901细胞过表达Flag-p27稳定细胞株是否构建成功。通过CCK-8药物敏感性实验检测p27在胃癌细胞顺铂耐药中所发挥的功能。结果:双酶切和测序结果表明，pCDNA 3.0-Flag-p27构建成功，并在293T中成功表达，胃癌SGC-7901细胞过表达Flag-p27细胞株建立成功，通过耐药曲线表明过表达p27可以引起SGC-7901细胞顺铂耐药。结论:成功构建了带Flag标签的p27真核表达载体，为进一步研究p27在胃癌耐药中的功能奠定了基础。

Construction and eukaryotic expression vector of p27 for cisplatin-resistance in SGC-7901

Objective:To construct the eukaryotic expression vector of p27 labeled with Flag tag and detect its function in cisplatin-resistance of SGC-7901.Methods:p27 gene was obtained by PCR from breast library and cloned into pCDNA 3.0-Flag vector.The recombinant plasmid was transfected into 293T cells and identified by either qRT-PCR or Western blot.Meanwhile，SGC-7901 stable cell line transfected with Flag-p27 was detected by Western blot.p27's function in cisplatin resistance of gastric cancer was studied by CCK-8 assay.Results:pCDNA3.0 Flag-p27 eukaryotic expression vector was successfully constructed by double digestion identification，and the inserted fragment was confirmed correct by sequencing.The expression of p27 in human 293T cells and SGC-7901 pCDNA3.0-Flag-p27 stable cell line were successfully identified.Furthermore，drug-resistance curve shows that Flag-p27 overexpression can cause the cisplatin-resistance in SGC-7901.Conclusion:The eukaryotic expression vector of pCDNA3.0 Flag-p27 was successfully constructed and expressed.This study laid a foundation for the further study of drug-resistance in gastric cancer.