促血管生成素2在胃癌中的表达及其反义基因转染对人胃癌细胞SGC-7901的影响

Expressions of angiopoietin2 in gastric carcinoma and effects of antisense angiopoietin2 cDNA on human gastric carcinoma cell line SGC-7901

摘要:

目的：探讨促血管生成素2（angiopoietin2，Ang2）在胃癌组织中的表达及其与肿瘤病理分期的联系；观察反义 Ang2基因转染对人胃癌细胞SGC-7901的影响。方法：用Western blot法检测Ang2蛋白在10例胃癌组织及10例正常胃黏膜组织中的表达，用免疫组化法检测53例不同病理分期的胃癌组织中Ang2蛋白的表达；应用脂质体转染法将既往实验合成[1]的 pEGFP-N1-anti Ang2转染体外培养的人胃癌细胞SGC-7901，以空载体（pEGFP-N1）转染组和未转染组作对照，RT-PCR及免疫组化检测转染后Ang2基因及蛋白的表达，MTT法和流氏细胞仪（flow cytometry，FCM）检测反义Ang2基因转染对细胞生长和细胞凋亡的影响；分别用上述3组细胞注射于裸鼠皮下，对比观察肿瘤生成的速度和肿瘤组织中的血管生成数量。结果：Ang2蛋白在正常胃黏膜组织中不表达，在不同病理分期的胃癌组织中均呈阳性表达（F=166.76，P＜0.000 1）；RT-PCR及免疫组化显示反义Ang２基因转染组无Ang2 mRNA及Ang2蛋白的表达，MTT法检测显示反义Ang2基因转染组细胞体外增殖能力明显低于其他两组（F=10.39，P=0.002 4），FCM检测显示其活细胞比率明显低于其他两组（χ2=3 188.980 5，P＜0.000 1）；转染了反义Ang2基因的胃癌细胞成瘤速度较其它两组明显减慢[第6天（F=10.18，P=0.000 5）、第18天（F=7.8０，P=0.002 1）及第30天（F=79.58，P＜0.000 1）]，肿瘤组织中血管生成的数量较其它两组明显减少[第1周（F=15.18，P＜0.000 1）、第2周（F=3.50，P=0.044 4）、第3周（F=39.02，P＜0.000 1）]。结论：胃癌组织中Ang2的表达与胃癌血管生成及胃癌侵袭能力密切相关；反义Ang2基因转染可封闭人胃癌细胞SGC-7901中Ang2 mRNA及Ang2蛋白的表达，抑制其体外生长，促进其凋亡；并对其体外成瘤性及其新生血管的生成有明显的抑制作用。

Abstract:

Objective：To study the expressions of angiopoietin2（Ang2） in gastric carcinoma tissues and its relationship with pathologi－cal stage and to investigate the biological effects of antisense Ang2 cDNA（pEGFP-N1-anti-Ang2 cDNA） on human gastric carcinoma cell line SGC-7901. Methods：Immunohistochemical method was used to detect expressions of Ang2 protein in 53 samples of gastric carcinoma tissues and Western blot was used to detect expressions of Ang2 protein in 10 samples of gastric carcinoma and 10 normal gastric tissues. Two different plasmids including pEGFP-N1 and pEGFP-N1-antisense Ang2 cDNA were transferred seperately into an in vitro cultured human gastric carcinoma cell line SGC-7901 by using Lipofectamine2000 transfection technique. mRNA and protein expressions of Ang2 after transfection in 53 samples of gastric carcinoma tissue were determined by RT-PCR and immunohisto－chemical method. Effects of antisense Ang2 cDNA on cell viability and apoptosis were determined by MTT assay and flow cytometry（FCM）. The three kinds of gastric carcinoma cells were subcutaneously injected into 30 nude mice and were divided into three groups. Velocity of tumor formation and amount of angiogenesis in tumor tissues were detected. Results：Ang2 protein was positively expressed in gastric carcinoma tissues at different pathological stages（F=166.76，P＜0.000 1），while was unexpressed in normal gastric tissues. Based on RT-PCR and immunohistochemical method，SGC-7901 cell transfected antisense Ang2 gene did not express Ang2 mRNA and its protein. MTT assay demonstrated that the group transfected with antisense Ang2 gene had much lower proliferative capacity than other groups（F=10.39，P=0.002 4）. FCM demonstrated that the group transfected with antisense Ang2 gene also had an decreased cell viability than the other groups（ χ2=3 188.980 5，P＜0.000 1）. Tumor formation was slower in cell transfected with antisense Ang2 gene than in other groups（the 6th d：F=10.18，P=0.000 5；the 18th d：F=7.80，P=0.002 1；the 30th d：F=79.58，P＜0.000 1）. Average number of newly formed vessels was fewer in mice transfected with antisense Ang2 gene than in the other groups（the 1th week：F=15.18，P＜0.000 1；the 2nd week，F=3.50，P=0.044 4；the 3rd week，F=39.02，P＜0.000 1）. Conclusions：Ang2 plays an important role in the angiogenesis and tumor formation. mRNA and protein expression of Ang2 in SGC-7901 cells can be inhibited by antisense Ang2 gene. Antisense Ang2 gene can suppress the in vitro growth of human gastric carcinoma cells as well as the growth and angio－genesis in the human SGC-7901 gastric carcinoma.