血管生成素-1基因对人胃癌细胞增殖与凋亡的影响

Effect of angiopoietin-1 gene on the proliferation and apoptosis of human gastric cancer cells

目的:探讨人血管生成素-1(angiopoietin-1．Angl)对胃癌细胞MGC-803增殖与凋亡的影响,研究其生物学作用及其在肿瘤发生中的作用机制．方法:以人胚肾293细胞对重组质粒Ad-Angl和Ad-GFP进行包装、扩增和纯化后转导MGC-803细胞,应用MTT比色法分析Angl对该胃癌细胞增殖的影响,通过流式细胞仪分析血浆Angl对胃癌细胞凋亡的影响,并通过免疫组织化学法检测Angl及其受体Tie-2的表达情况．结果:成功包装、扩增及纯化了重组腺病毒Ad-Angl及对照病毒Ad-GFP,其感染滴度分别为2．0×1012和1．15×1013PFU／L．当感染复数(MOI)为20时转导率即接近100％,而细胞形态无明显改变．MTT结果显示,24,48和72 h时Ad-Angl组吸光度均明显高于Ad-GFP组及对照组(Ad-GFP vs Ad-Angl:24 h,t=16．32, P<0．0001;48 h,t=10．47,P=0．0005;72 h,t= 24．59,P<0．0001．对照组vs Ad-Angl:24 h,t= 10．54,P=0．0005;48 h,t=13．92,P=0．0002; 72 h,t=22．46,P<0．0001),Ad-GFP组与对照组相比无差别:流式细胞仪检测显示,对照组、Ad-GFP组和Ad-Angl组的凋亡率(％)分别为: 9．90±0．62,10．07±1．27和4．17±0．35;免疫组化显示,3组细胞均有不同程度Ang-1及其受体Tie-2的表达,且其阳性表达率在Angl基因转染组(Ang-1:99．2±0．8;Tie-2:85．4±1．8)显著高于对照组(Ang-1:51．0±2．7;Tie-2:30．8±1．5)和GFP组(Ang-1:51．4±1．5;Tie-2:32．4±2．3)(P<0．000 1)．结论:Angl能显著促进人胃癌细胞MGC-803体外增殖,抑制血浆饥饿时的凋亡．

AIM： To explore the effect of angiopoietin-1 （Ang1） gene on the proliferation and apoptosis of human gastric cancer cell line MGC-803. METHODS： The replication-deficient adenovirus carrying human angiopoietin-1 （Ad-Ang1） and green fluorescent protein （Ad-GFP） were packaged, amplified and purified in human embryonic kidney cells 293 （HEK293）. Then gastric cancer cell line MGC-803 was transfected with the resultant adenoviruses. The proliferation and apoptosis of MGC-803 cells were measured by MTT colorimetry and flow cytometry, respectively. The expression of Ang1 and its receptor （Tie-2） were detected by immunohistochemistry. RESULTS： The recombinant adenovirus vector carrying Ang1 or GFP were amplified and purified successfully with the viral titers of 1.15 × 10^13 and 2.0×10^12 PFU/L, respectively. The transfection rate almost reached 100% when the multiplicity of infection （MOI） was 20. The proliferation of MGC-803 cells in Ad-Ang1-transfected group was markedly higher than that in Ad-GFP-transfected group （24 h： t = 16.32, P 〈 0.0001; 48 h： t = 10.47, P = 0.0005; 72 h： t = 24.59, P 〈 0.0001） and control group （24 h： t = 10.54, P = 0.0005; 48 h： t = 13.92, P = 0.0002; 72 h： t = 22.46, P 〈 0.0001）. The apoptotic rate of Ag-Ang1-transfected MGC-803 cells was obviously lower than that of Ag-GFP-transfected ones （t = 7.73, P = 0.0015）, but there was no difference between Ag-GFP group and control group. The positive rates of Ang1 and Tie-2 expression were significantly higher in Ad-Ang1-transfected cells （Ang-1： 99.2 ± 0.8; Tie-2：85.4 ± 1.8） than those in the control （Ang-1： 51.0 ± 2.7; Tie-2：30.8 ± 1.5） and Ag-GFP-transfected ones （Ang-1： 51.4 ± 1.5; Tie-2：32.4 ± 2.3）, respectively （P 〈 0.0001）. CONCLUSION： Ang1 gene may obviously promote the proliferation and inhibit the apoptosis of human gastric cancer cell line MGC-803.