次黄嘌呤-鸟嘌呤磷酸核糖转移酶缺陷型EL/4细胞系的建立  

Establishment of hypoxanthine guanine phosphoribosyltransferase deficient EL/4 cell line

作　　者：龚文[1] 禚娅[1] 蒋砚秋[1] 潘晓园[1] 邵启祥[1] 

机构地区：[1]江苏大学基础医学与医学技术学院免疫学研究室,江苏镇江212013

出　　处：《江苏大学学报（医学版）》2011年第4期285-288,共4页Journal of Jiangsu University：Medicine Edition

基　　金：国家自然科学基金资助项目(30671984);江苏省自然科学基金资助项目(BK2008231)

摘　　要：目的:建立稳定的次黄嘌呤-鸟嘌呤磷酸核糖转移酶(hypoxanthine guanine phosphoribosyltransferase,HG-PRT)缺陷型EL/4细胞系,为小鼠T细胞杂交瘤的制备奠定基础。方法:通过乙基甲磺酸(ethyl methanesulfonate,EMS)提高EL/4细胞的基础突变率,用8-氮杂鸟嘌呤(8-Azaguanine,8-AG)从5μg/ml到80μg/ml浓度逐渐筛选出对8-AG有稳定抗性的细胞株EL/4,并对其生物性状与基因表型进行鉴定。结果:通过筛选获得能够耐受高浓度(80μg/ml)8-AG的EL/4细胞,并能长期在20μg/ml 8-AG培养液中正常生长,但不能耐受次黄嘌呤-氨甲蝶呤-胸腺嘧啶(hypoxanthine-aminopterin-thymidine,HAT)培养基(3 d全部死亡),RT-PCR未能从该EL/4细胞中检测出HGPRT基因的表达,由此可见该EL/4细胞的HGPRT基因发生突变或者缺失。将该细胞经克隆后获得单克隆细胞株,并命名为突变的EL/4(mutant EL/4,mEL/4)。该细胞株能与磁珠分离的小鼠脾脏来源的CD4+T细胞在PEG作用下顺利进行融合。结论:成功建立了HGPRT缺陷型EL/4细胞系mEL/4,为制备T细胞杂交瘤奠定了基础。Objective： To establish a hypoxanthine guanine phosphoribosyl-transferase（HGPRT） deficient EL/4 cell line for setting up mouse T cell subset hybridoma.Methods： After treated with ethyl methanesulfonate（EMS）,the EL/4 cells were selected by 8-Azaguanine（8-AG） form lower concentration（5 μg/ml） to higher concentration（80 μg/ml） step by step.The mRNA expression of HGPRT of the resistant cells was detected by RT-PCR.In the end,8-AG selected EL/4 cells were tested for fusing with CD4＋T lymphocytes isolated from mouse spleen by Dynabeads.Result： The selected EL/4 cells could resistant with higher concentration（80 μg/ml） of 8-AG and maintained in 20 μg/ml 8-AG medium,and couldn′t survive within hypoxanthine-aminopterin-thymidine（HTA） medium.The result of RT-PCR revealed that there was no mRNA expression of HGPRT gene can be detected in these 8-AG resistant cells,and the cell line was named as mutant EL/4 cells（mEL/4）.Furthermore,FCM analysis shown that mEL/4 cells could be fused with the CD4＋T lymphocytes isolated from mouse spleen.Conclusion： The HGPRT deficient EL/4 cell line（mEL/4） was established successfully,it will provide useful cell line for establishing T cell hybridoma.

关 键 词：乙基甲磺酸 8-氮杂鸟嘌呤 次黄嘌呤-鸟嘌呤磷酸核糖转移酶 突变 EL/4淋巴瘤细胞 

分 类 号：R393[医药卫生—基础医学]