丝裂霉素C对制备干细胞饲养层效果的影响  

Effect of mytomycin-C on preparetion of stem cell feeder layer

作　　者：彭三凤 王利[1] 周学亮[1] 陈美娟[1] 陈东阳[1] 杨小淦[1] 许慧艳[1] 卢晟盛[1] 张明[1] 卢克焕[1] 陆阳清[1] 

机构地区：[1]广西大学亚热带农业生物资源保护与利用国家重点实验室/广西高校动物繁殖与动物生物技术重点实验室,南宁530005

出　　处：《南方农业学报》2015年第5期905-910,共6页Journal of Southern Agriculture

基　　金：广西科学研究与技术开发计划项目(桂科合1347004-6);广西高等学校高水平创新团队及卓越学者计划项目(2014)

摘　　要：【目的】筛选出丝裂霉素C制备干细胞饲养层的最佳处理浓度，为建立完善干细胞饲养层培养体系奠定基础。【方法】以STO（sIM小鼠成纤维细胞耐硫代鸟嘌呤和耐鸟苯苷亚系）、MEF（原代分离的小鼠胎儿成纤维细胞）和BRL（大鼠肝细胞）为研究对象，分别以0、2．5、5．0、10．0、15．0、20．0μg／mL丝裂霉素C处理汇合度为90％～100％的STO、MEF、BRL细胞单层2h，继续培养3d后统计各处理组饲养层细胞的存活数量及增殖细胞比例，并用EdU免疫荧光标记法检测细胞的增殖状况。【结果】随着丝裂霉素C处理浓度的增加，饲养层增殖细胞数量占总细胞数量的比例均呈逐渐降低趋势。丝裂霉素C处理STO、BRL、MEF的最佳浓度分别为5．0、10．0和10．0Izg／mL，对应的增殖细胞比例分别为6．8％、3．O％和2．1％。【结论】丝裂霉素c能有效抑制干细胞饲养层细胞的增殖并保持良好活性，但不同饲养层细胞的最佳处理浓度有所差异。[Objective]The present experiment was conducted to screen optimum concentration of mitomycin-C to pre- pare stem cell feeder layer, in order to lay the foundation for establishing and perfecting culture system of embryonic stem cell feed layer. [ Method ]Using STO （SIM mouse enbryo-derived thiognanine and ouabain resistant） cells, MEF （mouse embryonic fibroblasts） cells and BRL （buffalo rat liver） cells as materials, these cells with the confluence of 90%-100% were treated with mytomyein-C at 0, 2.5, 5.0, 10.0, 15.0 and 20.0 ~g/mL for 2 h, respectively, then which were cul- tured sequentially in medium without mytomyein-C for 3 days. Finally, the survival number and proliferation percentage of each treatment were calculated statistically, and the proliferation of feeder layer cell was detected by using EdU im- munofluorescence. [Result]The results showed that, the ratio of proliferous ceils to total ceils number decreased gradually with the increase of mitomyein-C concentration. Therefore, the optimum concentrations of mytomyein-C with for STO, BRL and MEF cells were 5.0, 10.0 and 10.0 ~g/mL, respectively, the corresponding proliferous cell ratio was 6.8%, 3.0% and 2.1%, respectively. [Conclusion]The mitomyein-C can effectively inhibit feeder layer cell from proliferating and keep its good activity. While there are some differences in optimum concentration of mitomycin-C among ceils be- tween different feeder layer.

关 键 词：饲养层 丝裂霉素C EdU免疫荧光检测 细胞增殖 

分 类 号：Q813.11[生物学—生物工程]