文章摘要
陈 蓉,余 辉,安艳丽.表达内皮抑素基因的内皮祖细胞治疗肝癌的初步研究[J].南京医科大学学报,2016,(10):1181~1187
表达内皮抑素基因的内皮祖细胞治疗肝癌的初步研究
Study of endostatin gene transfected mice bone marrow-derived endothelial progenitor cells for liver carcinoma
投稿时间:2016-05-31  
DOI:10.7655/NYDXBNS20161007
中文关键词: 内皮祖细胞  内皮抑素  肝癌
英文关键词: endothelial progenitor cells  endostatin
基金项目:国家自然科学基金青年基金(81201131);江苏省自然科学基金青年基金(BK2012335)
作者单位
陈 蓉 东南大学附属中大医院肿瘤科,江苏 南京 210009 
余 辉 江苏省肿瘤医院介入科,江苏 南京 210009 
安艳丽 东南大学医学院,江苏省分子影像与功能影像重点实验室,江苏 南京 210009 
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中文摘要:
      目的:观察内皮抑素(endostatin,ES)基因转染的内皮祖细胞(endothelial progenitor cells,EPCs)对小鼠肝癌细胞H22增殖的影响,探讨联合自体EPCs和ES基因治疗肝癌的可行性和有效性。方法:构建表达ES基因的慢病毒载体,培养小鼠骨髓源EPCs,运用qPCR检测培养内皮细胞表面标记CD31?血管内皮生长因子受体(vascular endothelial growth factor receptor,VEGFR)?血管性血友病因子(von Willebrand factor,vWF)表达,电镜观察内皮细胞特征小体。实验分EPCs组?EPCs+LV空病毒载体组?EPCs+ES组。CCK-8法检测各组细胞增殖的情况。同时构建原位肝癌小鼠模型,尾静脉注射3组细胞后不同时间点处死小鼠,测量肿瘤大小并进行统计分析。结果:①酶切和测序?PCR鉴定证实成功构建慢病毒载体pLenti6.3-ES-Monomer-DsRed;②qPCR显示培养细胞表达内皮细胞标志CD31?VEGFR?vWF,透射电镜下在细胞质内可见多个散在内皮细胞特征性Weibel-Palade小体(WP小体);③慢病毒载体Lenti6.3-ES-Monomer-DsRed转染小鼠骨髓源性EPCs后荧光显微镜下可见细胞呈红色荧光;EPCs+ES组细胞上清较对照组上清液对小鼠肝癌细胞H22体外增殖具有明显抑制作用,体内实验显示EPCs+ ES组肝癌生长速度慢于对照组。结论:小鼠骨髓源单个核细胞体外可以诱导培养为内皮祖细胞,负载ES基因的EPCs体外可以抑制小鼠肝癌细胞H22的增殖,体内可以抑制小鼠肝癌生长。
英文摘要:
      Objective:To study the endostatin(ES) gene armed endothelial progenitor cells(EPCs) for liver carcinoma H22 of mice and to discuss the feasibility and effectiveness of autologous EPSs combined with ES for treawent of liver carcinoma. Methods:We construct a lentiviral vector expressing endostatin gene and cultured the bone-marrow derived EPCs. Cell surface markers were detected with qPCR and cells were observed by electron microscope. There were three groups:EPCs,EPCs+LV,and EPCs+ES. Cell enhancement was detected by CCK-8 assay in the three groups. Liver carcinoma models in situ were made and three groups of cells were injected through tail veins. Mice were sacrificed later at different times and the tumors were detected. Results:The construction of lentiviral vectors pLenti6.3-ES-Monomer-DsRed was confirmed by cleavage,sequence identification and PCR. The cultured cells expressed endothelial cells’ markers CD31,VEGFR and VWF with qPCR. Characteristic WP globule for endothelial cell was observed by transmission electron microscope. EPCs transfected with ES were observed red. Supernatant in the group of EPC+ES could inhibit the enhancement of H22 than the group of EPC and EPC+LV. In vivo assay showed that the tumors of EPCs+ES were smaller than the other two groups. Conclusion:Mice bone marrow derived MNCs can be induced to EPCs,EPCs transfected with ES could supress the enhancement of H22 in vitro and the liver carcinoma in vivo.
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