反义促血管生成素2基因稳定转染对人胃癌细胞SGC-7901体外生长的抑制作用

Growth suppression of human gastric carcinoma cell line SGC-7901 by stable transfection of antisense angiopoietin2 gene

摘要:

目的：观察以pEGFP-N1为载体的促血管生成素2（angiopoietin2，Ang2）反义基因转染的人胃癌细胞SGC-7901中Ang2蛋白的表达，研究其对人胃癌细胞体外生长的抑制作用。方法：应用重组质粒转染反义Ang2的pEGFP-N1载体（pEGFP-N1-anti Ang2）、pEGFP-N1空载体质粒以脂质体转染法转染体外培养的人胃癌细胞株，以未转染组为对照，经G418筛选抗性克隆，扩大培养后，应用RT-PCR技术与免疫组化法检测Ang2基因及其蛋白的表达，MTT法分析细胞生长抑制作用，Annexin V-FITC和PI双染流式细胞仪检测细胞凋亡。结果：RT-PCR与免疫组化均示pEGFP-N1-anti Ang2转染组无Ang2 mRNA及其Ang2蛋白的表达，MTT检测表明pEGFP-N1-anti Ang2转染组活细胞数较其它两组均低，差异有统计学意义（F=10.39，P=0.002 4），流式细胞仪检测则显示其凋亡率较其它两组明显增高，差异有统计学意义（?字2=3 188.98，P＜0.000 1）。结论：pEGFP-N1-anti Ang2成功转染人胃癌细胞SGC-7901且可封闭Ang2 mRNA表达，使已转染pEGFP-N1-anti Ang2的人胃癌细胞Ang2蛋白的表达减少，并抑制人胃癌细胞的恶性表型。

Abstract:

Objective：To evaluate expressions of angiopoietin2（Ang2） protein in human gastric cancer cells SGC-7901 transfected by pEPFP-N1-antisense Ang2 and to investigate whether antisense Ang2 gene could inhibit the vitro growth of human gastric carcinoma cell line. Methods：Two different plasmids including pEGFP-N1 and pEGFP-N1 SGC-7901 antisense Ang2 were separately trans－ferred into an in vitro cultured human gastric cancer cell line SGC-7901 using Lipofectamine2000 transfection technique. After trans－fection，the cells were selected by G418. Then resistant clones were chosen and expanded in DMEM culture medium，with parental SGC-7901 cells as control. RT-PCR and immunohistochemical method were done to determine whether target genes and its proteins had expressed. Cell viability was determined by MTT assay. Apoptosis was determined by flow cytometry with a double dyeing method using FITC-conjugated annexin V and PI. Results：According to results of RT-PCR and immunohistochemical methods，angiopoietin mRNA and its proteins were not expressed in transfected antisense Ang2 gene SGC-7901 cells. MTT assay showed that pEGFP-N1-anti Ang2 transfection group had much lower cell viability than other groups（F=10.39，P=0.002 4）. Meanwhile，pEGFP-N1-anti Ang2 transfection group had an increase in apoptosis rates compared with those of the other groups based on results of flow cytometry（?字2=3 188.98，P＜0.000 1）. Conclusions：pEGFP-N1-anti Ang2 successfully transfects human gastric cancer cells SGC-7901，which in－hibits Ang2 mRNA and protein expressions as well as suppresses the malignant phenotype of human gastric cells.