本文選題：人淋巴細(xì)胞　切入點(diǎn)：HepG2細(xì)胞　出處：《內(nèi)蒙古大學(xué)》2011年碩士論文

【摘要】：本研究用化學(xué)誘變劑N-甲基-N'-硝基-N-亞硝基胍(MNNG)誘變建立次黃嘌呤鳥嘌呤磷酸核糖轉(zhuǎn)移酶(HGPRT)缺陷型的HepG2細(xì)胞與Hela細(xì)胞系,并將其與人扁桃體淋巴細(xì)胞進(jìn)行細(xì)胞融合,得到兩種HGPRT缺陷型細(xì)胞和雜交細(xì)胞。 對(duì)手術(shù)切除的扁桃體進(jìn)行淋巴細(xì)胞分離,得到混合淋巴細(xì)胞,尼龍棉柱分離T、B淋巴細(xì)胞,并分別用E-花環(huán)法與ELISA法鑒定細(xì)胞。E-花環(huán)形成百分比為59.4%, ELISA鑒定出細(xì)胞表面CD23抗原濃度在30 pg/ml-60 pg/ml之間。由扁桃體分離得到的混合淋巴細(xì)胞顯微鏡下觀察,細(xì)胞呈規(guī)則的圓型,且透亮,體外培養(yǎng)4-5天出現(xiàn)聚集生長(zhǎng)現(xiàn)象。尼龍棉柱分離的T淋巴細(xì)胞、B淋巴細(xì)胞在外形上沒有明顯的差別。B淋巴細(xì)胞于體外培養(yǎng)第7天進(jìn)入衰亡期,后便迅速死亡,第9天時(shí)全部死亡。T淋巴細(xì)胞可在體外連續(xù)培養(yǎng)20天,但隨后也進(jìn)入衰亡期,逐漸死亡。 通過N-甲基-N'-硝基-N-亞硝基胍(MNNG)誘導(dǎo)HepG2細(xì)胞與Hela細(xì)胞,逐步提高培養(yǎng)液中6-巰基鳥嘌呤(6-TG)的濃度,篩選出對(duì)6-TG有抗性的細(xì)胞株,檢測(cè)它在HAT中生長(zhǎng)的情況,得到可在含20μg/ml 6-TG的高糖DMEM完全培養(yǎng)基中穩(wěn)定生長(zhǎng)的細(xì)胞株,此細(xì)胞株在HAT培養(yǎng)基中第三天出現(xiàn)明顯的死亡,第12天全部死亡,誘變后細(xì)胞的染色體分別分布于32-88、36-86,染色體眾數(shù)分別為48、68。突變篩選出的細(xì)胞具有對(duì)6-TG抗性和對(duì)HAT敏感的特性,證實(shí)該細(xì)胞是HGPRT缺陷型細(xì)胞株,為以后進(jìn)行雜交瘤制備提供了適宜的親本細(xì)胞。 將新鮮分離的人扁桃體淋巴細(xì)胞與處于對(duì)數(shù)生長(zhǎng)期的HGPRT缺陷型HepG2細(xì)胞(HepG2-)和缺陷型Hela細(xì)胞(Hela-)在PEG的作用下分別進(jìn)行融合,經(jīng)過HAT選擇培養(yǎng)基的篩選、克隆分別得到淋巴細(xì)胞-HepG2雜交細(xì)胞與淋巴細(xì)胞-Hela雜交細(xì)胞。雜交瘤細(xì)胞的獲得使人淋巴細(xì)胞可以在體外長(zhǎng)期培養(yǎng),為體外產(chǎn)生人源性單克隆抗體的研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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