摘要
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 Objectives Stably transfecting the Secretory eukaryotic expression vector of human canstatin gene into CHO-K1 cells to get the cell strain that can express the canstatin proteins stably and study the biologic action of canstatin. Methods Stably transfect the Secretory eukaryotic expression vector of human canstatin gene into CHO-K1 cells to get the cell strain that can express the canstatin proteins stably and identify it by RT-PCR assay and Western-blotting assay. Then study more biologic actions of canstatin by more assays. Results Secretory eukaryotic expression vector of recombined human canstatin gene was successfully transfected CHO-K1μcells stably and it was approved that the cell strain can express the canstatin proteins stably. In vivo, the supernatant collected from the culture medium of CHO-K1/pSecTag2B/canstatin cells could significantly inhibit angiogenesis in CAM. The supernatant collected from the culture medium of CHO-K1/pSecTag2B/canstatin cells also showed that it can inhibit the growth of the HUVEC-12 and induce its apoptosis. Conclusion 1. Secretory eukaryotic expression vector of human canstatin gene was successfully transfected into CHO-K1 cells by positive ion liposome method, and the cell strain which can express the canstatin proteins stably is acquired. 2. The supernatant collected from the culture medium of CHO-K1/pSecTag2B/canstatin cells possesses the biological activity of inhibiting angiogenesis in chick embryo chorioallantoic membrane and it can inhibit the growth of the HUVEC-12 and induce apoptosis of it.
出处
《中国实用医药》
2007年第15期4-8,共5页
China Practical Medicine
基金
湖南省卫生厅课题(编号:Y02-068)
关键词
稳定转染
脂质体
鸡胚绒毛尿囊膜实验
生长曲线
流式细胞学技术
steady transfection
Liposome
Chick embryo chorioallantoic membrane(CAM) assay
Curve of growth
Flow cytometry
