外源血小板衍生生长因子(PDGF)B链基因在CHO细胞的表达

本文总结应用DNA-磷酸钙盐沉淀的基因转移技术,把含人c-sis cDNA的质粒pSM-1,以单独转染或与pSV_2neo共转染方法转入CHO细胞(中国仓鼠卵巢细胞),经低血清或G_(418)筛选分离得到多个有PDGF表达的细胞株。其中PDGF高表达件——FB_6,细胞形态和生长行为明显改变,可在软琼脂培基上形成集落,细胞生长速率加快,能在低血清(2%)培液中长期传代,细胞的条件培液有刺激静止的NRK细胞(大鼠肾成纤维细胞)DNA合成的活力。RNA点杂交和Southern Blot显示FB_5细胞有??PDGF mRNA的高表达和人c-sis基因的整合,而且,在连续传代7个月后,FB_5细胞基因组中仍然有人c-sis基因存在,说明CHO细胞的不正常生长和转化是由于PDGF基因的稳定整合和高表达所引起。这一稳定转化株(FB_5)是进一步研究PDGF对细胞生长控制和转化功能的理想模型。

EXPRESSION OF EXOGENOUS PLATELET-DERIVED GROWTH FACTOR B CHAIN GENE IN CHO CELLS

CHO cells were transfected with plasmidpSM-1 (containing human c-sis cDNA) sin-gly or co-transfected with pSV 2 neo DNAby calcium phosphate method.After lowserum or G418 selection several cell lineswith expression of platelet-derived growthfactor (PDGF) were obtained.One amongthem,FB_5,was of the highest PDGF expre-ssion and showed the following biologicalcharecteristics when compared with CHO cells:(1) a prominant change in morphology fromspindle to round in shape;(2) increase ofgrowth rate;(3) growth in low serum (2%)medium as a semisuspension culture;(4)growth on soft agar to larger colonies;(5)synthesis of PDGF in cytoplasm identified byimmunofluorescent method;(6) the condition-ed medium stimulated DNA synthesis ofNRK cells;(7) RNA dot hybridization show-ing high transcription of PDGF mRNA;(8) southern blot showing integration ofhuman c-sis gene was still stable after 7months. These results indicated that intergrationof exogenous c-sis gene and its high expres-sion might cause CHO cells to high growthrate and even transformation.The establish-ment of this stable transformed cell line,FB_5is thought to be a good model for furtherstudy on the function of PDGF in cell grow-th control and cell transformation.