细胞培养实验中条件培养基相关问题探讨  

Study on the conditioned medium in vitro experiments

作　　者：赵志敏[1] 叶永安[1] 王硕仁[1] 朱陵群[1] 高永红[1] 孙逸坤[1] 

机构地区：[1]北京中医药大学东直门医院,北京100700

出　　处：《中西医结合肝病杂志》2010年第1期23-26,共4页Chinese Journal of Integrated Traditional and Western Medicine on Liver Diseases

基　　金：全国重点高校博士点课题(No.20060026010)

摘　　要：目的:通过LX-2人肝星状细胞条件培养基(CM)对HepG2人肝癌细胞增殖活性的影响,探讨CM各影响因素在离体实验中的作用。方法:收集LX-2细胞第24小时、48小时、72小时条件培养基,设100%、50%、25%共3个浓度,并设1%DMEM组为对照,培养24小时、48小时、72小时;并将收集的第24小时条件培养基分装分别保存至4℃1周、-20℃1周、-80℃3个月、-20℃3个月,采用MTT法检测各组条件培养基对HepG2细胞增殖活性的影响。结果:①收集时间:第24小时、第48小时LX-2CM具有明显的促增殖作用(P<0.01);培养24小时后,各收集时间点的LX-2CM的促增殖作用为:第24小时>第48小时>第72小时(P<0.01);培养48小时后,第48小时>第72小时(P<0.01)。②添加浓度:培养24小时后,第24小时CM100%浓度、第24小时CM50%浓度、第24小时CM25%浓度、第48小时CM100%浓度均有明显的促增殖作用(P<0.01);第24小时CM各浓度比较,100%优于50%(P<0.05);第48小时CM各浓度,100%优于50%(P<0.05)、25%(P<0.01)。③多元线性回归分析,条件培养基收集时间与HepG2细胞增殖呈负相关,浓度与其呈正相关。④保存条件:-20℃1周CM优于-80℃3个月CM、-20℃3个月CM(P<0.01);-80℃3个月CM优于-20℃3个月CM(P<0.01)。结论:LX-2CM对HepG2细胞具有明显的促进增殖活性作用,其作用随着收集时间点延长、浓度减小,增殖效果逐渐降低;CM的保存条件对其作用效果有影响。Objective： To observe the effect of conditioned medium （CM） of LX-2 human-derived hepatic stellate cell on proliferation in HepG2 cells, to explore the roles of conditioned medium with different factors in vitro experiment. Methods： The conditioned medium of LX-2 were collected after 24, 48 and 72hs, and divided into three groups in three concentration levels, which were 100%, 50%, 25%, 1% DMEM as the control group. The conditioned medium of LX-2 collected after 24hs were divided into 4 groups according to preservation conditions, which were 4~C one week, - 20℃ one week, - 80℃ three months, - 20℃ three months, and the influence of the preservation conditions on the conditioned medium were observed. MTT colorimettic assay was used to observe the influence on proliferation of HepG2 cells after incubate with different CM. Results： （1）CM collecting time points： contrast with 1% DMEM group, the groups of CM collected on 24h and 48h had significantly proliferation on HepG2 cells, and the differences were statistical significance （ P 〈 0. 01 ） . The differences among the groups of CM collected on 24h, 48h and 72h after incubated for 24h were statistical significance （P 〈0. 01 ） . After incubated for 48h, The differences between the groups of CM collected on 48h and 72h were statistical significance （ P 〈 0. 01 ） . （2）The added concentration of the CM ： contrast with 1% DMEM group, three concentration levels in 24h group and the concentration level of 100% in 48h group had significantly proliferation, and the differences were statistical significance. Among the three concentration levels in 24h group, the differences between 100% and 50% were statistical significance, so did in 48h group. （3）multiple linear regression analysis： there was a significant negative correlation between the collecting time points of the CM and the proliferation of HepG2. While there was a positive correlation between the concentration levels of the CM and the proliferation. （4）The preserva

关 键 词：条件培养基 收集时间点 浓度 细胞增殖 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]