HGPRT缺陷型细胞系建立及其与人淋巴细胞的融合

发布时间：2018-04-10 05:23

本文选题：人淋巴细胞　切入点：HepG2细胞　出处：《内蒙古大学》2011年硕士论文

【摘要】：本研究用化学诱变剂N-甲基-N'-硝基-N-亚硝基胍(MNNG)诱变建立次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT)缺陷型的HepG2细胞与Hela细胞系,并将其与人扁桃体淋巴细胞进行细胞融合,得到两种HGPRT缺陷型细胞和杂交细胞。对手术切除的扁桃体进行淋巴细胞分离,得到混合淋巴细胞,尼龙棉柱分离T、B淋巴细胞,并分别用E-花环法与ELISA法鉴定细胞。E-花环形成百分比为59.4%, ELISA鉴定出细胞表面CD23抗原浓度在30 pg/ml-60 pg/ml之间。由扁桃体分离得到的混合淋巴细胞显微镜下观察,细胞呈规则的圆型,且透亮,体外培养4-5天出现聚集生长现象。尼龙棉柱分离的T淋巴细胞、B淋巴细胞在外形上没有明显的差别。B淋巴细胞于体外培养第7天进入衰亡期,后便迅速死亡,第9天时全部死亡。T淋巴细胞可在体外连续培养20天,但随后也进入衰亡期,逐渐死亡。通过N-甲基-N'-硝基-N-亚硝基胍(MNNG)诱导HepG2细胞与Hela细胞,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20μg/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培养基中第三天出现明显的死亡,第12天全部死亡,诱变后细胞的染色体分别分布于32-88、36-86,染色体众数分别为48、68。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。将新鲜分离的人扁桃体淋巴细胞与处于对数生长期的HGPRT缺陷型HepG2细胞(HepG2-)和缺陷型Hela细胞(Hela-)在PEG的作用下分别进行融合,经过HAT选择培养基的筛选、克隆分别得到淋巴细胞-HepG2杂交细胞与淋巴细胞-Hela杂交细胞。杂交瘤细胞的获得使人淋巴细胞可以在体外长期培养,为体外产生人源性单克隆抗体的研究奠定了基础。
[Abstract]:In this study, HepG2 cells and Hela cell lines deficient in Hypoxanthine guanosine phosphotransferase (HGP) were established by mutagenesis of chemical mutagens N- methyl-N- (N-nitro-N-nitrosoguanidine), and fused with human tonsil lymphocytes.Two types of HGPRT deficient cells and hybrid cells were obtained.Lymphocytes were separated from the tonsil removed by surgery and mixed lymphocytes were obtained. The T _ (B) lymphocytes were separated by nylon cotton column.The percentage of cell. E- rosette formation was 59.4 by E- rosette method and ELISA method respectively. The concentration of CD23 antigen on cell surface was identified by ELISA in the range of 30 pg/ml-60 pg/ml.The mixed lymphocytes isolated from tonsils were observed under microscope. The cells were regular round and bright. The aggregation and growth appeared in vitro for 4-5 days.There was no obvious difference in the appearance of T lymphocyte B lymphocytes isolated from nylon cotton column. B lymphocytes entered the decaying stage on the 7th day of culture in vitro, and then died rapidly.On the 9th day, all the T lymphocytes could be cultured continuously for 20 days in vitro, but then they also entered the stage of death and died gradually.HepG2 cells and Hela cells were induced by N- methyl-Na-nitro-N-nitro-nitro-guanidine (MNNGs), and the concentration of 6-mercaptoguanine 6-TG in the culture medium was gradually increased. The cell lines resistant to 6-TG were screened out, and their growth in HAT was detected.The cell line which could grow stably in the high sugar DMEM complete medium containing 20 渭 g/ml 6-TG was obtained. The cell line died obviously on the third day and died on the 12th day in the HAT medium.The chromosomes of the mutated cells were distributed in 36-86, and the chromosome motifs were 48.68 respectively.The mutant cells were resistant to 6-TG and sensitive to HAT. It was confirmed that the cells were HGPRT deficient cell lines, which provided a suitable parent cell for the preparation of hybridoma in the future.The newly isolated human tonsil lymphocytes were fused with HGPRT deficient HepG2 cells HepG2-and defective Hela cells in logarithmic growth phase under the action of PEG. The selected medium was selected by HAT.Lymphocyte-HepG2 hybrid cells and lymphocyte-Hela hybrid cells were cloned respectively.The acquisition of hybridoma cells allows human lymphocytes to be cultured for a long time in vitro, which lays a foundation for the production of human monoclonal antibodies in vitro.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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