Ang-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

The expression of angiopoinet-2 in gastric carcinoma and the effects of transfected by pEGFP-N1-anti-Ang-2 cDNA on human gastric carcinoma cell line SGC-7901

摘要:

目的：探讨促血管生成素-2(Angiopoietin-2，Ang-2)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 Ang-2基因转染对人胃癌细胞 SGC-7901的影响。方法：用 Western Blot法检测 Ang-2蛋白在 10例胃癌组织及 10例正常胃组织中的表达，用免疫组化法检测 53例不同病理分期的胃癌组织中 Ang-2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 Ang-2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 Ang-2基因及蛋白的表达， MTT法和流氏细胞术检测反义 Ang-2基因转染对细胞生长和细胞调亡的影响；分别用上述三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：Ang-2蛋白在正常胃组织中不表达，在胃癌组织中呈阳性表达，并且随病理分期进展表达呈增强趋势；RT-PCR及免疫组化显示反义Ang-2基因转染组无 Ang-2 mRNA及 Ang-2蛋白的表达，MTT法检测显示反义 Ang-2基因转染组活细胞数较其它两组均低（P＜0.05），流氏细胞术检测显示其调亡率较其它两组明显增高（P＜0.01）；转染了反义 Ang-2基因的胃癌细胞成瘤速度较其它两组明显减慢（P＜0.05），肿瘤组织中血管生成的数量较其它两组明显减少（P＜0.05）。结论：胃癌组织中 Ang-2的表达与胃癌血管生成及胃癌侵袭能力密切相关；反义 Ang-2基因转染可封闭人胃癌细胞 SGC-7901中 Ang-2 mRNA及 Ang-2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 expression of Ang-2 in gastric carcinoma tissue, and its relation with pathological stage, To investigate the biological effects of antisense Ang-2 cDNA （pEGFP-N1-anti-Ang-2 cDNA）in human SGC-7901 gastric carcinoma cell line. Methods: Immunohistochemical method was used to detect the expression of Ang-2 protein in 53 samples of gastric carcinoma tissues, and western blot technique was used to the expression of Ang-2 protein in 10 samples of gastric carcinoma tissues and 10 normal gastric tissues. Using the lipofectamine 2000 transfection technique,We transferred separately two different plasmids induding pEGFP-N1,pEGFP-N1- antisense Ang-2 cDNA into an in vitro cultured human gastric carcinoma cell line SGC-7901. The expression of Ang-2 mRNA in 53 gastric carcinoma tissue specimens were determined by RT-PCR. Immunohistochemistry was used to detect the expression of Ang-2 in those tissues as well.The cell viability was detrmined by MTT assay.Apoptosis was determined by flow cytometry . the three kinds of gastric carcinoma cells were subcutaneously injected into 30 nude mice divided into three groups freely. After injection, the tumor mass grew and the angiogenesis of the tumor could be detected. Results:Ang-2 was expressed in gastric carcinoma tissue, while normal gastric tissue was unexpressed. An increased expression of Ang-2 was strong association between the different pathological stages of tumors. On RT-PCR and immunohistochemical method,the SGC-7901 cells transfected antisense Ang-2 gene did not show expression of Ang-2 mRNA and its protein.MTT assay showed the group transfected with antisense Ang-2 gene had a much lower cell viability than other groups(P＜0.05).The group also had an increase in the rate of apoptosis as measured by flow cytometry Compared with other groups(P＜0.01). the tumor grew slower in transfected mice with antisense Ang-2 gene than other groups. The average number of newly formed vessels in the transfected mice with antisense Ang-2 gene was smaller than other groups（P＜0.05）. Conclusions:Ang-2 plays an important role in the angiogenesis and tumor growth, the expression of Ang-2 mRNA and its protein in SGC-7901 cells can be inhibited by antisense Ang-2 gene.The study also show that antisense Ang-2 gene can suppress the in vitro growth of human gastric carcinoma cells. The antisense Ang-2 gene may suppress growth and angiogenesis in the human SGC-7901 gastric carcinoma apparently.