新基因ANGPTL4的克隆及其在血管新生中的功能研究

Cloning of a novel gene, ANGPTL4 and the functional study in angiogenesis

目的 克隆新基因ANGPTL4 ,并探讨其功能。方法 采用以细胞生长为基础的功能筛选方法筛选人胎盘cDNA文库 ,结合RACE PCR方法克隆到一个人全长新基因ANGPTL4 ,用RH PCR方法对其进行染色体定位 ;用原核表达系统对重组ANGPTL4进行蛋白表达 ;用四甲基偶氮唑蓝 (MTT)试验、原代猪主动脉内皮细胞 (PAEC)体外迁移试验、PAEC体外浸润试验、PAEC体外管状试验及小鼠体内栓子形成试验探讨了新基因ANGPTL4对体内外血管新生过程的影响。结果 获取的人全长基因ANGPTL4定位于染色体 19p13 3,其原核表达产物对PAEC的体外增殖没有明显作用 (P =0 0 5 04 ) ,但ANGPTL4的 2 μg/ml剂量组对PAEC的体外迁移和浸润均有明显的促进作用 (P <0 0 5 ) ,对其管状结构形成也有程度不同的促进作用 ;对小鼠体内血管新生有较明显的促进作用。

Objective To clone a novel HCC related gene and study its functions. Methods Using a combination of cell growth based functional screening of human placental cDNA library and RACE, a novel gene, angiopoietin like 4 (ANGPTL4) was isolated. The gene was mapped by RH PCR; also it was expressed with E. coli. Effects on angiogenesis was studied by MTT assay, in vitro wound migration assay, in vitro invasion assay, in vitro tube formation assay and in vivo mouse matrigel plug assay. Results ANGPTL4 was cloned and mapped in 19p13.3. ANGPTL4 had no significant effect on proliferation of PAEC ( P =0.050 4), while it can significantly stimulate migration and invasion of PAEC with the dose of 2 μg/ml ( P <0.05) and promote in vitro tube formation of PAEC and angiogenesis of mouse in vivo. Conclusions ANGPTL4 can stimulate angiogenesis.