血管抑素对胰腺癌血管生成的抑制作用

Pancreatic cancer cell inhibition and anti-angiogenesis by angiostatin in vivoand in vitro

目的观察血管抑素在胰腺癌血管生成中的作用。方法采用Lipofectamine 2000基因转染技术将真核表达载体pcDNA3.1(+)-angiostatin导入人胰腺癌PC-3细胞,筛选阳性克隆并扩大培养。PC-3细胞分为血管抑素转染组、空白对照组及脂质体对照组,分别检测各组血管抑素蛋白表达;利用显微镜下细胞计数法测定转染前后PC-3细胞的体外生长曲线;检测各组PC-3细胞培养上清所分泌的血管抑素对血管内皮细胞ECV-304增殖的影响。进一步建立种植瘤模型,通过CD34染色法检测Angiostatin对种植瘤中血管密度的影响。结果 Western blotting检测显示血管抑素转染组的PC-3细胞可分泌血管抑素,而空白对照组及脂质体对照组的PC-3细胞无血管抑素的分泌;细胞生长曲线显示,血管抑素转染组、空白对照组及脂质体对照组的细胞生长速度和倍增时间无明显差异;3组PC-3细胞上清液对内皮细胞ECV-304的生长有明显差异,血管抑素转染组的生长明显低于空白对照组及脂质体对照组。体内试验显示血管抑素转染组的种植瘤生长明显低于空白对照组及脂质体对照组。血管抑素转染组的种植瘤微血管密度(19.6±3.6)显著少于空白对照组(48.5±4.7)和脂质体对照组(51.1±5.4)。结论血管抑素可抑制血管内皮细胞的增殖,进一步产生抑制胰腺癌血管生成效应。

Objective To observe the inhibition of pancreatic cancer cells with anti-angiogenesis by angiostatin in vitro and in vivo.Methods The recombinant vector pcDNA3.1(+)-angiostatin was transfected into human pancreatic cancer cells PC-3 with lipofectamine 2000.Angiostatin protein expression was determined by Western blot.The supernatant was collected to treat endothelial cells and cell proliferation in vitro was observed under microscope.The size of tumors was measured,and microvessel density count(MVD) in tumor tissues was assessed by immunohistochemistry with primary anti-CD34 antibody.Results After transfected into PC-3 with lipofectamine 2000 and selected by G418,macroscopic resistant cell clones were formed in the experiment group transfected with pcDNA 3.1(+)-angiostatin and vector control group.After treatment with the supernatant,the endothelial cell(ECV-304) proliferation was inhibited.In nude mice model,markedly inhibited tumorigenesis and slowed tumor expansion were observed in the experiment group as compared to those in the control group.The microvessel density was obviously smaller in the experiment group(19.6±3.6) than in the blank control group(48.5±4.7) and the liposome control group(51.1±5.4).Conclusion Angiostatin inhibits the proliferation of endothelial cell growth in vitro and further exerts an anti-tumor function through antiangiogenesis in a paracrine way in vivo.