内皮抑素基因重组腺病毒载体的构建及其对新生血管的影响

Construction of adenovirus-mediated mouse Endostatin and study on its inhibitory effect on angiogenesis

目的构建腺病毒介导的小鼠内皮抑素并观察其在体内、外的表达及对血管的抑制作用。方法以mEndostatin质粒为模板通过PCR方法扩增回收mEndostatin基因,将mEndostatin基因连接到腺病毒载体DC315中,构建PDC315-mEndo表达质粒。将此表达质粒与腺病毒重组质粒pBGHE3通过lipofectamine 2000共同转染293细胞,经同源重组产生重组腺病毒Ad-mEndo,用氯化铯密度梯度超速离心法纯化。用50%组织培养感染剂量法测定病毒滴度。体外转染骨肉瘤细胞株MG-63,用western印迹检测感染的骨肉瘤细胞上清夜mEndostatin的表达并通过鸡胚绒毛尿囊膜实验(chorioallantoic membrane,CAM)观察其抑制血管生成的活性。结果酶切、PCR及DNA测序鉴定表明成功构建了携带内皮抑素(Endostatin,ES)融合基因的重组腺病毒载体,而在鸡胚绒毛尿囊膜血管生成实验中加入培养物上清的实验组与对照组相比鸡胚尿囊膜血管密度稀疏,平均血管数差异明显(P<0.01)。结论腺病毒介导的小鼠内皮抑素基因在体外获得高效表达,并可显著抑制血管生成。

Objective To construct a adenovirus-mediated mouse endostatin （Ad-mEndo）to observe its expression in vivo and in vitro, and study its inhibitory effect on angiogenesis. Methods Use mEndostatin plasmid as template to amplificate and recover mEndostatin gene by PCR, and then connect mEndostatin gene to adenovirus vector PDC315 to construct PDC315-mEndo plasmid. Recombinant adenovirus plasmid PDC315-mEndo was cotransfected with pBGHE3 into 293 packaging cells by lipofectamine 2000, and then homologously recombine the recombinant adenovirus Ad-mEndo. After that, the recombinant adenovirus was purified by cesium chloride density gradient ultracentrifugation, and the titer was measured by 50% tissue culture infective dose. Osteosarcoma cells（ MG-63 ）were infected with Ad-mEndo in vitro, then the infected osteogenic sarcoma cell supematant was detected by Western blot to observe the expression of mEndostatin and its inhibitory effects of angiogenesis by chorioallantoic membrane （CAM） experimentation. Results The recombinant adenovirus vector was constructed successfully Which was proved by enzyme digestion,PCR and DNA sequencing, and the CAM blood vessel of test-group added with culture supernatant were less than that in control-group （ P 〈 0. 01 ）. Conclusion Adenovirus-mediated mouse Endostatin is constructed successfully and is expressed effectively in vitro with a significant inhibitory effect on angiogenesis.