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目的将人canstatin cDNA分泌型真核表达载体pSecTag2B/canstatin稳定转染中国仓鼠卵巢(CHO-K1)细胞并获得稳定表达canstatin蛋白产物的细胞株。再进一步研究表达产物的生物学作用。方法将人canstatin cDNA的重组1质粒pSecTag2B/canstatin稳定转染CHO-K1细胞,应用RT-PCR、western-blotting法检测表达产物,再用多种方法进一步研究canstatin的生物学作用。结果成功的将pSecTag2B/canstatin稳定转染CHO-K1细胞并鉴定出上清液中有目的蛋白的表达。该细胞培养上清液能抑制鸡胚绒毛尿囊膜中微血管的生成,抑制人脐静脉内皮细胞(HUVEC-12)的生长并诱导其凋亡。结论①成功的将pSecTag2B/canstatin稳定转染CHO-K1细胞并获得能稳定表达can-statin蛋白产物的细胞株;②证实含canstatin蛋白产物的细胞培养上清液具有抑制鸡胚绒毛尿囊膜微血管生成的活性并可在体外抑制HUVEC-12的生长和诱导其凋亡。
Objective To stably transfected Chinese hamster ovary (CHO-K1) cells with human canstatin cDNA secreting eukaryotic expression vector pSecTag2B / canstatin and to obtain cell lines stably expressing canstatin protein. Further study of the biological role of expression products. Methods The recombinant plasmid pSecTag2B / canstatin containing human canstatin cDNA was stably transfected into CHO-K1 cells. The expression products were detected by RT-PCR and western-blotting. The biological effects of canstatin were further studied by a variety of methods. Results The pSecTag2B / canstatin was successfully transfected into CHO-K1 cells and the expression of the target protein in the supernatant was identified. The cell culture supernatant can inhibit the formation of microvasculature in chick chorioallantoic membrane, inhibit the growth of human umbilical vein endothelial cells (HUVEC-12) and induce apoptosis. CONCLUSIONS ①PstTag2B / canstatin was stably transfected into CHO-K1 cells successfully and cell lines stably expressing can-statin protein product were obtained. ② Cell culture supernatants containing canstatin protein product were confirmed to have inhibitory effects on chick chorioallantoic membrane microvascular Generated activity and can inhibit the growth of HUVEC-12 and induce its apoptosis in vitro.