不同细胞培养基对PAM212细胞产生TSLP的影响

Effect of Different Cell Media on TSLP Expression in PAM212 Cells

比较MEMα、RPMI1640、DMEM和EMEM 4种细胞培养基中PAM212细胞产生胸腺基质淋巴细胞生成素(TSLP)的不同,为研究PAM212细胞产生TSLP的作用机制提供适宜的细胞培养基。采用ELISA法测定培养上清中TSLP的蛋白水平,MTT法检测PAM212细胞的存活率。无刺激时,PAM212细胞在培养基DMEM和EMEM中产生的TSLP是MEMα和RPMI1640中的5～6倍,但是药物刺激后TSLP的增加倍率在MEMα和RPMI1640中更高。同样,细胞生存率基本不变时,02F04、肿瘤坏死因子-α(TNF-α)或12-邻-14-烷酰佛波醇-13-乙酸酯(TPA)刺激PAM212后虽然能在DMEM中产生更多的TSLP,但是在MEMα中诱导的TSLP增加倍率更高。PAM212细胞产生TSLP的水平与其从播种开始使用的培养基直接相关,而与复苏及播种前使用的培养基关系不大;刺激前无论哪个时间点停止添加10%FBS于细胞培养液中,PAM212细胞产生的TSLP均显著减少,且停止添加FBS的时间越长,细胞存活率降低越多。总之,PAM212在不同细胞培养基中产生TSLP的水平不同。如果想无刺激时PAM212细胞能产生较高水平的TSLP,选用DMEM培养基较为适宜;如果想研究药物诱导TSLP产生的作用机制,选用刺激后TSLP增加倍率高的MEMα培养基较为适宜。

To provide suitable cell culture media for the study of thymic stromal lymphopoietin(TSLP)production in PAM212 cells by comparing TSLP levels in four different cell culture media,TSLP levels in culture supernatant and cell viability were determined by ELISA and MTT assay.TSLP level in media of Dulbecco’s modified Eagle’s medium(DMEM)or Eagle’s minimum essential medium(EMEM)was 5~6 times higher than that in Minimum essential medium alpha medium(MEMα)or RPMI1640,but the increase of TSLP expression in media of MEMαor RPMI1640 was higher than that in DMEM or EMEM.In order to investigate the effect of medium change on TSLP production,cell culture media were changed firstly,and then the cells were seeded,cultured for24 h,and stimulated by 02F04 for 24 h.In order to compare TSLP production induced by the same stimuli in different cell culture media,PAM212 cells were seeded directly,cultured for 24 h,and stimulated by 02F04 at 10μmol/L,TNF-αat 30 ng/mL or 12-Otetradecanoylphorbol 13-acetate(TPA)at 3 nmol/L for 24 h,respectively.The results showed that TSLP production in PAM212 cells was directly related to the medium which was used from seeding,but not to that used in cell resuscitation or before seeding.TSLP levels induced by the three inducers were higher in the medium of DMEM,but the increase in TSLP expression was higher in MEMα.Besides,when PAM212 cells were cultured with fetal bovine serum(FBS)-free DMEM 10-h before or from the treatment of02F04,TSLP production was significantly decreased,and the longer time FBS was removed for,the lower cell viability PAM212 cells had.Taken together,the levels of TSLP produced by PAM212 cells in various cell culture media were different.The medium of DMEM is suitable for a study in which a high level of TSLP in PAM212 cells without stimuli is required,whereas MEMαis suitable for the study on the mechanism of some stimuli on TSLP production in PAM212 cells because TSLP expression can be induced to a higher level.