人肝癌细胞系HepG2的次黄嘌呤鸟嘌呤磷酸核糖转移酶缺陷型的建立

摘要

本试验旨在建立次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)缺陷型的HepG2细胞系,为细胞杂交与单克隆抗体的研究提供亲本细胞.通过N-甲基-N-硝基-N-亚硝基胍(MNNG)诱导HepG2细胞突变,逐步提高培养液中6-巯基鸟嘌呤(6-TG)的浓度,筛选出对6-TG有抗性的细胞株,检测它在HAT中生长的情况.得到可在含20 μg/mL 6-TG的高糖DMEM完全培养基中稳定生长的细胞株,此细胞株在HAT培养基中第3天出现明显的死亡,第12天全部死亡,诱变后细胞的染色体分布于32-88,染色体众数为48.突变筛选出的细胞具有对6-TG抗性和对HAT敏感的特性,证实该细胞是HGPRT缺陷型细胞株,为以后进行杂交瘤制备提供了适宜的亲本细胞.%To establish a hypoxanthine guanine phosphoribosyl transferase(HGPRT) deficient cell line from HepG2 cell line,so as to provide parent cells for hybridoma and monoclonal antibody study. The HepG2 cells were cultured with N-methylN-nitro-N-nitrosoguanidine (MNNG) for mutation and selected by increasing concent ration of 6-thioguanine (6-TG). The 6-TG resistant mutant cells were cultured in the medium containing 6-TG or HAT. After four months of screening,a deficient cell line was established. It could grow vigorously in the medium containing 20 μg/mL 6-TG, while it all died within 12 days in the medium containing HAT. Karyotypic analysis showed that the number of the chromosomes of the deficient cells is between 32 and 88, with the modal number being 48. The 6-TG resistant and aminopterin-sensitive character of the mutant cell line reveals that it is deficient in HGPRT. The cell line established in this study will be a good parent cell line for preparation of hybridoma in the future.