血管抑素K1-5腺病毒载体的构建及对人血管内皮细胞的抑制作用

Construction of adenovirus vector with angiostatin K1-5 gene and suppression to proliferation and migration of human vascular endothelial cells

目的构建携带血管抑素的腺病毒载体,并探讨其对血管内皮细胞增殖及迁移的抑制作用。方法采用基因重组技术,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。扩增纯化后,病毒滴度达1.1×1010pfu/mL。实验表明,携带血管抑素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,且能使血管内皮细胞趋化能力减弱,抑制血管形成。结论成功构建了携带血管抑素K1-5的腺病毒载体,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。

Objective To construct adenovirus vector with agiostatinK1-5 gene and to investigate the function of suppression to proliferation and migration for vascular endothelial cells. Methods With the use of gene recombination and clone technology, we constructed the adenovirus vector with the gene of angiostatin K1-5. In vitro vascular endothelial eclls proliferation assay and migration activity were performed through direct infection, MTI＂ and transwell chemotaxis assay. Results 50% TCID indicated that the condence of resultant viruses was 1.5 ×10^9pFU/mL. It was purified by CsCL banding,final yield were generally 1.1 × 10^10 PFU/mL plaquing-forming units. Through indirect infect assay and MTT, we found angiostatin K1-5 inhibited human vascular endothelial ceils 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 significantly inhibited HUVEC migration. Conclusion We successfully constructed adenoviruse vector with angiostatin K 1 - 5 and demonstrated its inhibitory effect on proliferation and migration of HUVEC.