基础医学与临床 ›› 2008, Vol. 28 ›› Issue (4): 370-375.

• 研究论文 • 上一篇    下一篇

血管抑素K1-5腺病毒载体的构建及对血管内皮细胞的抑制作用

刘恩令 周玉秀 糜若然 钱其军   

  1. 河北医科大学附属唐山市工人医院妇产科 河北医科大学附属唐山市工人医院妇产科 天津医科大学总医院妇产科 上海市第二军医大学东方肝胆外科医院病毒基因治疗室
  • 收稿日期:2007-03-14 修回日期:2005-06-27 出版日期:2008-04-25 发布日期:2008-04-25
  • 通讯作者: 刘恩令

Construction of adenovirus vector with angiostatink1-5 gene and the study of the function of suppression to proliferation and migration for human vascular endothelial cells

En-ling LIU, Yu-xiu ZHOU, Ruo-ran MI, Qi-jun QIAN   

  1. Tang Shan Worker Hospital,He Bei Medical University
  • Received:2007-03-14 Revised:2005-06-27 Online:2008-04-25 Published:2008-04-25
  • Contact: En-ling LIU,

摘要: 目的 构建携带血管抑素的腺病毒载体,并对其对血管内皮细胞增殖及迁移的抑制作用进行研究。 方法 采用基因重组及克隆技术,构建携带血管抑素K1-5的腺病毒载体,PCR鉴定,50%组织培养感染剂量法测定病毒滴度及氯化铯密度梯度离心法纯化病毒。直接感染台盼蓝染色排除法、MTT及体外趋化实验检测血管抑素K1-5对内皮细胞增殖及趋化的抑制作用。 结果 经50%组织培养感染剂量法测定病毒滴度及氯化铯密度梯度离心法纯化病毒,病毒滴度为1.5×109pfu/ml。按需要扩增一定量后,进行纯化,病毒滴度达1.1×1010pfu/ml。直接感染台盼蓝染色排除法及MTT法检测表明,血管抑素K1-5的腺病毒能抑制血管内皮细胞增生,并对其具有杀伤力。趋化实验表明,血管抑素K1-5能使血管内皮细胞趋化能力减弱,抑制血管形成。 结论 我们成功构建了携带血管抑素K1-5的腺病毒载体,并经直接感染台盼蓝染色排除法、MTT及体外趋化实验证明,血管抑素K1-5能明显抑制血管内皮细胞增殖及迁移,为进一步体内实验,研究对血管形成的抑制作用奠定了基础。

Abstract: Objective To construct adenovirus vector with agiostatink1-5 gene and investigate the function of suppression to proliferation and migration for vascular endothelial cells.Methods With the use of gene recombination and clone technology ,we construct the adenovirus vector with the gene of angiostatin k1-5. Ad-angiostatin k1-5 was isolated from a single plaque,expanded in 293 cells and purified by cesium chloride contrifugation. In vitro vascular endothelial eclls proliferation assay and migration activity were investigated through direct infection,MTT and transwell chemotaxis assay. Results 50%TCID indicated that the condence of resultant viruses was 1.5×109PFU/mL. It was purified by CsCL banding,final yield were generally 1.1×1010 PFU/mL plaquing-forming units. Through indirect infect assay and MTT, we found angiostatin k1-5 could inhibit human vascular endothelial cells proliferation. We utilized human vascular endothelial cells to study the effect angiostatin k1-5 on cell migration ,the result showed that adenoviruse vector with angiostatin k1-5 could significantly inhibited HUVEC migration .Conclusions: We successfully constructed adenoviruse vector with angiostatin k1-5 and showed in vitro it could inhibit proliferation and migration of HUVEC.