内皮抑制素相互作用蛋白HT036基因的全长克隆及其促血管上皮细胞凋亡的分子机制

目的：克隆与内皮抑制素（endostatin）相互作用蛋白HT036基因的全长，进一步验证其与内皮抑制素的相互作用，初步阐明其促血管上皮细胞凋亡的分子机制。 方法：实验于2004—10／2006-03在解放军总医院呼吸科实验室、军事医学科学院放射医学和生物工程研究所实验室完成。①根据已知HT036的EST序列设计引物。应用RACE技术，从人胎肝cDNA文库获取该基因全长。②采用免疫共沉淀和Western Blot验证HT036蛋白与内皮抑制索在真核细胞内存在相互作用。③利用真核瞬时表达载体pCDNA3．1（＋）在EVC304细胞中瞬时表达HT036蛋白，转染了HT036基因的EVC304细胞为试验组（重复两次）；没有转染HT036基因的细胞为对照组，用流式细胞仪检测HT036基因对细胞凋亡和细胞周期的影响。 结果：①HT036基因全长克隆及生物信息学分析结果：利用RACE技术成功获得HT036基因的全长，并在GENEBANK进行了注册，注册号AY775560。②免疫共沉淀和Western—blot的验证结果：证实HT036蛋白能与内皮抑制素在真核细胞中相互作用。③HT036基因对细胞凋亡和细胞周期的影响：成功构建了表达HT036基因的真核表达载体，两次试验转染ECV304细胞后24h的凋亡率平均为23．63％，而对照组仅为0．10％。 结论：获得了一与内皮抑制紊相互作用的新的蛋白HT036基因的全长，初步表明HT036参与了内皮抑制索调节的血管生成过程，并对血管上皮细胞具有促进凋亡的作用。

Full-length clone of protein HT036 gene interacting with endostatin and its molecular mechanism in promoting the apoptosis of vascular endothelial cell

AIM： To clone the full length of protein gene HT036 interacts with endostatin, and further identify the interaction between them and elucidate the molecular mechanism of HT036 gene in promoting the apoptosis of vascular endothelial cells.
METHODS： The experiment was conducted in the Laboratory of Respiration of General Hospital of Chinese PLA and the Laboratory of Radiology and Bioengineering of Academy of Military Medical Sciences between October 2004 and March 2006, （1） RACE was used to clone the full length of HT036 gene from human liver cDNA library based on its known EST sequence. （2） Co-immunoprecipitation technology and Western Blot were adopted to identify the interaction between HT036 protein and endostatin in eucaryotic cell. （3） The eucaryotic transient expression vector pCDNA3.1 （＋） which expressed HT036 protein was constructed and transferred into EVC304 cells. The EVC304 cells being transferred into HT036 were taken as the experiment group （repeated twice）, and EVC30-4 cells without transfection were taken as the control group. Flow cytometry was used to detect the effect of HT036 gene on cell apoptosis and cell cycles.
RESULTS： （1） Analysis of the full length of HT036 gene clone and its bioinformatics： The full length of HT036 was successfully cloned and registered into Genebank with the number of AY775560. （2） Co-immunoprecipitation and Western-blot proved that HT036 protein could interact with endothelial protein in COS-7 cells. （3） Effect of HT036 on cell apoptosis and cell cycle： HT036 gene was successfully inserted into pCDNA3.1 vector and transferred into cell EVC304 twice with an average apoptotic rate of 23.63% at 24 hours after transfection, while that in the control group was just 0.10%.
CONCLUSION： The full length of HT036 gene that interact with the endostatin is obtained, and it is primarily manifested that HT036 participates in the vascularization which is regulated by endostain and can promote the apoptosis of vascular endothelial c