血管形成抑制因子HIAF-1的克隆及其抗肿瘤作用

Cloning and antitumor activity of angiogenesis inhibitor HIAF-1

目的 克隆和表达一种新的血管形成抑制因子 ,并研究其抗肿瘤活性。方法 应用逆转录聚合酶链反应从人胎儿肝脏组织中克隆到一种新的血管形成抑制因子 (humaninhibitingangiogenesisfactor 1,HIAF 1) ,经测序后克隆到表达载体pET30a(+)上 ,在E .coliBL2 1∶DE3菌中表达。用MTT法体外检测HIAF 1对内皮细胞的作用 ,采用小鼠乳癌自发性肺转移模型IVTA2MA 891,体内研究重组蛋白对肿瘤血管形成的作用和抑瘤作用。结果 从胎儿肝脏组织中首次克隆到人源血管形成抑制因子HIAF 1,经测序为人胶原ⅩⅧC 末端的一部分。在E .coli细胞中高效表达了该因子的重组蛋白 ,蛋白表达量为 88mg/L。体外实验表明HIAF 1能抑制内皮细胞的增殖 ,IC50 值为 7.5 μg/ml。动物实验表明该重组蛋白能够显著抑制肿瘤血管形成 ,原发瘤的生长 (抑瘤率为 46 .6 % )及肿瘤的转移 (抑制转移率为 6 8.9% )。结论 成功的克隆了血管形成抑制因子HIAF 1,其能够抑制肿瘤血管的形成 ,在肿瘤的治疗中显示出良好的应用前景。

Objective A new angiogenesis inhibitor HIAF 1 (human inhibitor angiogenesis factor 1) was cloned, expressed in E. coli and its antitumor activity was studied. Methods HIAF 1 was amplified by RT PCR from human fetal liver tissue,then cloned into pET30a(+) vector and expressed in E.coli BL21:DE3 after sequencing. In vitro endothelial proliferation inhibiting activity of HIAF 1 was examined by MTT method. In vivo antitumor activity was studied in a murine model of IVTA2MA 891. Results HIAF 1 was first cloned from human fetal liver tissue. Sequence analysis of the inhibitor revealed identity to a c terminal fragment of human collagen XVIII. HIAF 1 was effectively expressed in E. coli with a yield of 88 mg/L. Recombinant HIAF 1 protein could inhibit endothelial cell proliferation in vitro with an IC 50 value of 7.5 μg/ml. In vivo studies showed that HIAF 1 inhibited growth rate of the primary tumor by 46.6% and metastasis by 68.9%.Conclusion The cloned and the bio engineering product of HIAF 1 is an angiogenesis inhibitor,capable of inhibiting tumor growth and metastasis.