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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q PCR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,v WF)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p Lenti6.3-ES-Monomer-Ds Red;(2)q PCR显示培养细胞表达内皮细胞标志CD31、VEGFR、v WF,透射电镜下在细胞质内可见多个散在内皮细胞特征性WeibelPalade小体(WP小体);(3)慢病毒载体Lenti6.3-ES-Monomer-Ds Red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。
OBJECTIVE: To investigate the effect of endothelial progenitor cells (EPCs) transfected with endostatin (ES) gene on the proliferation of mouse hepatocellular carcinoma cell line H22 and to explore the feasibility and effectiveness of the combination of autologous EPCs and ES genes in the treatment of liver cancer . Methods: The lentiviral vector expressing ES gene was constructed and the mouse bone marrow-derived EPCs were cultured. Q-PCR was used to detect the expression of CD31, vascular endothelial growth factor receptor (VEGFR), von Willebrand’s disease Von Willebrand factor (v WF) expression, endothelial cells observed by electron microscopy. The experimental sub-EPCs group, EPCs + LV empty vector group, EPCs + ES group. CCK-8 method to detect the proliferation of cells in each group. At the same time, the model of orthotopic liver cancer was established. Three groups of cells were injected into tail vein to kill mice at different time points. The size of tumor was measured and analyzed statistically. Results: (1) The lentiviral vector p Lenti6.3-ES-Monomer-Ds Red was successfully constructed by restriction enzyme digestion and sequencing. (2) q PCR showed that the cultured cells expressed endothelial markers CD31, VEGFR, Under the electron microscope, a number of scattered WeibelPalade bodies (WP bodies) were observed in the cytoplasm. (3) Lentiviral vector Lenti6.3-ES-Monomer-Ds Red transfected mouse bone marrow-derived EPCs under fluorescence microscope The cells showed red fluorescence. The supernatant of EPCs + ES group had a significant inhibitory effect on the proliferation of mouse hepatoma cell line H22 in vitro. The in vivo experiments showed that the growth of hepatocellular carcinoma in EPCs + ES group was slower than that in control group. CONCLUSION: Mouse bone marrow-derived mononuclear cells can be induced to culture endothelial progenitor cells in vitro. ESCs loaded with ESCs can inhibit the proliferation of mouse hepatoma H22 in vitro and inhibit the growth of mouse hepatocellular carcinoma in vivo.