Hela细胞HGPRT缺陷型细胞系构建  

Establishment and Identification of HGPRT Deficient Hela Cell Line

作　　者：郭惠东[1] 特日格乐[1] 尚爱萍[1] 郑文静[1] 刘刚[1] 道日娜[1] 王蓉蓉[1] 李瑶[1] 李煜[1] 

机构地区：[1]内蒙古大学哺乳动物生殖生物学与生物技术教育部重点实验室,呼和浩特010021

出　　处：《中国细胞生物学学报》2013年第2期170-174,共5页Chinese Journal of Cell Biology

基　　金：国家大学生创新性实验计划项目(批准号:101012618)资助的课题~~

摘　　要：建立稳定的次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)缺陷的Hela细胞系,为细胞融合相关研究和人源化单克隆抗体制备提供有利于筛选的亲本细胞。通过诱变剂N-甲基-N′-硝基-N-亚硝基胍(MNNG)对Hela细胞进行诱变,逐步提高培养基中6-巯基鸟嘌呤(6-TG)的浓度,筛选出对6-TG稳定耐受的细胞,在次黄嘌呤-氨基喋呤-胸腺嘧啶(hypoxanthine-aminopterin-thymidine,HAT)培养基中鉴定其敏感性,最后对筛选得到的Hela-HGPRT-进行生物学鉴定。在此基础上,将Hela-HGPRT-细胞系与人淋巴细胞融合,在HAT培养基中筛选杂交细胞。筛选得到了能够长期在含20μg/mL 6-TG培养基中生长的Hela-HGPRT-细胞,并且在HAT培养基中不能存活。Hela-HGPRT-细胞与人淋巴细胞成功融合,获得能够连续传代培养的杂交瘤细胞。经MNNG诱导和6-TG筛选,得到了稳定传代的Hela-HGPRT-细胞系,该细胞系可用于细胞融合相关研究。To establish an HGPRT- deficient Hela cell line for creation of hybridoma to produce humanized monoclonal antibody. Mutation of Hela cells was induced with MNNG and selected in gradient concentrations of 6-TG. Then, the sensitivity of mutant cells to HAT medium was tested. After the stable Hela-HGPRT- cells were developed, they were fused with human B lymphocytes and selected in HAT medium.Hela-HGPRT- cells could survive in medium containing 20 μg/mL 6-TG in the long term and could not live in HAT medium. We also succeeded in fusing human B lymphocytes with Hela-HGPRT- cells and the hybridoma cells could be continually cultured. Using the methods of induction with MNNG and screening with 6-TG, we obtained a stable Hela-HGPRT- cell line and it could be continually cultured. This Hela-HGPRT- cells could be used in the research of cell fusion.

关 键 词：HELA细胞 HGPRT缺陷 MNNG 6-TG 细胞融合 

分 类 号：R737.33[医药卫生—肿瘤]