体外构建内皮抑素基因真核表达载体抑制血管生成

Construction of pcDNA3-Endo eukaryon expression plasmid and angiogenesis inhibition in vitro

背景:胶质瘤的根除治疗至今仍是一大难题,抗血管生成治疗胶质瘤有望成为新的有效途径。目的:证实内皮抑素在体外对血管生长的抑制作用,为今后利用其抑制肿瘤生长奠定实验室基础。方法:取Wistar大鼠肝脏,提取mRNA后利用RT-PCR获取内皮抑素cDNA片段。碱裂解法小量提取质粒pcDNA3。重组质粒pcDNA3-Endo的构建。重组pcDNA3-Endo真核表达载体转染骨髓间充质干细胞。RT-PCR及Western Blot检测内皮抑素基因的表达。MTT法检测ECV-304细胞增殖抑制实验。体外实验共分成4个组:重组质粒组、空载质粒组、脂质体对照组及空白对照组。结果与结论:成功构建pcDNA3-Endo重组真核表达质粒,pcDNA3-Endo质粒能在体外有效转录并分泌内皮抑素基因,转染了外源pcDNA3-Endo质粒的ECV-304细胞增殖明显受到抑制。结果提示内皮抑素基因能在体外有效抑制血管内皮细胞的增殖。

BACKGROUND： The eradication therapy of glioma is the major problem, and anti-angiogenesis therapy is a potential treatment of glioma. OBJECTIVE： To confirm the inhibiting effect of endostatin on angiogenesis in vitro, and to lay the foundation in inhibiting the growth of tumor by endostatin in the future. METHODS： Endostatin mRNA was extracted from the liver of Wistar rats by Trizol and endostatin cDNA was synthesized by RT-PCR. Endostatin cDNA and pcDNA3 were connected and pcDNA3-Endo racombined plasmid was constructed successfully. The recombinant pcDNA3-Endo was transfected into bone marrow mesenchymal stem cells by Lipofectamine. The expression of endostatin was identified by RT-PCR and western blot analysis. Endostatin proteinum activity was detected by ECV-304 cell proliferation inhibition experiment using MTT assay. The in vitro experiments were divided into four groups： recombinant plasmid group, vector plasmid group, liposome control group and blank control group. RESULTS AND CONCLUSION： PcDNA3-Endo eukaryon expression plasmid was constructed successfully. Endostatin gene can be transcribed and expressed effectively in vitro by pcDNA3-Endo plasmid. The growth of ECV-304 cell was inhibited obviously by pcDNA3-Endo. The growth of vascular endothelial cells can be inhibited obviously by endostatin gene.