稳定表达rhPDGF-BB的CHO细胞系的建立及其生物学特性研究  

Study on Establishment of CHO Cell Line with Stable Expression of rhPDGF-BB and Its Biological Character

作　　者：董荣娜[1] 毋中明[1] 宋丹[1] 张绍敏[1] 李兰英[1] 于德民[1] 

机构地区：[1]天津医科大学代谢病医院,卫生部激素与发育重点实验室,300070

出　　处：《天津医药》2009年第2期124-127,共4页Tianjin Medical Journal

基　　金：中国博士后科学基金项目(项目编号:20070410766);天津卫生局科技基金项目(项目编号:07kz47)

摘　　要：目的:建立稳定表达人血小板衍化生长因子-BB(PDGF-BB)全长的中国仓鼠卵巢细胞(CHO)细胞系,并检测表达产物的生物活性。方法:通过脂质体瞬间转染的方法将含有rhPDGF-BB全长的质粒转染CHO细胞系。利用抗生素筛选的方法使转染阳性的细胞扩增和单克隆化,进一步稳定传代。通过Western印迹法挑选高表达克隆,进而使用MTT法检测转染前后细胞增殖活性和转染细胞上清培养NIH3T3细胞的增殖情况。反复传代、冻存和复苏鉴定表达的稳定性。结果:获得稳定且高效表达rhPDGF-BB的CHO细胞系;转染前后细胞生长速度存在差异,NIH3T3细胞的增殖随转染细胞上清的浓度和培养时间的增加而增加。结论:pSELECT-GFPzeo-MCS载体能作为稳定表达的载体用于筛选。稳定表达rhPDGF-BB的CHO细胞具有新的生物学功能,实现了组织工程种子细胞的构建,为研究糖尿病溃疡创面愈合的生物材料提供实验基础。Objective： To establish a CHO cell line that stably expresses wild type platelet-derived growth factor-BB （PDGF-BB）, and to investigate the basic biological activities of the expression products. Methods： CHO cell line was transfected by liposome with a plasmid containing whole length PDGF-BB, and positive clones were selected by antibiotic. Western blotting was used to examine the level of rhPDGF-BB expression, and the proliferation of the cell line was determined by MTT. MTT method was used to detect the proliferation of NIH 3T3 cell after it was cultured in the supernatant of these CHO cells. Repeated passage, freezing and resuscitating were used to identify the stability of the PDGF-BB expression. Results： The CHO cell line that stably expressed rhPDGF-BB was obtained, and there was a difference of cell proliferation between pre-and post-transfection, the proliferation of NIH 3T3 cell increased with the increasing of supernatant concentration and culture time. Conclusion： The vector pSELECT-GFPzeo-MCS can be used with antibiotic selection to establish stable cell line. The CHO cell line that stably expressed rhPDGF-BB has a new biological character, and successfully constructs the seed ceils for tissue engineering, which can afford experiment base and theory foundation to study the biomaterial for the ulcer concrescence.

关 键 词：血小板源性生长因子 CHO细胞 基因 转染 皮肤溃疡 糖尿病并发症 

分 类 号：R392.11[医药卫生—免疫学] R734.2[医药卫生—基础医学]