人脐带间充质干细胞体外培养过程中的表观遗传学改变  

The epigenetic alterations of human umbilical cord mesenchymal stem cells during in vitro culture

作　　者：张宪毅[1,2] 李强 潘莹莹 侯艳 李煜[1] 郭明洲[4] 叶颖 

机构地区：[1]内蒙古大学生命科学学院,内蒙古呼和浩特010021 [2]内蒙古干细胞医学工程技术研究中心 [3]北京京蒙高科干细胞技术有限公司 [4]中国人民解放军总医院消化科

出　　处：《胃肠病学和肝病学杂志》2012年第6期506-509,共4页Chinese Journal of Gastroenterology and Hepatology

基　　金：内蒙古自然科学基金资助项目(2011BS0507)

摘　　要：目的分离、培养人脐带间充质干细胞(hUC-MSCs),并探讨体外培养过程中表观遗传学的改变。方法随机选取足月顺产健康胎儿脐带,采取复合酶消化法从脐带胶质中获得hUC-MSCs。流式细胞术检测细胞表面标志物。分化培养基诱导hUC-MSCs分化。β-半乳糖苷酶染色检测细胞衰老。甲基化特异性PCR(MSP)检测hUC-MSCs在不同培养时期特定基因的甲基化状态。蛋白免疫印记(Western blot)检测hUC-MSCs在不同培养时期MGMT基因的蛋白表达。结果按整根脐带30 cm计算,可收获(3～6)×106hUC-MSCs。培养的hUC-MSCs高表达CD29、CD73、CD105、CD90和CD44,不表达CD45、CD34和HLA-DR。hUC-MSCs可定向分化为成脂、成骨和成软骨细胞。随着细胞的传代,衰老细胞占细胞总数的比例增加。培养后期的hUC-MSCs出现MGMT基因甲基化。结论复合酶消化法可从脐带胶质中获得具有间充质干细胞(MSC)特性的细胞。随着传代次数的增多,DNA修复基因MGMT出现甲基化,可能参与hUC-MSCs衰老调控。Objective To isolate and culture human umbilical cord mesenchymal stem cells （hUC-MSCs） and to study the epigenetic alterations during in vitro culture. Methods hUC-MSCs were separated from Warton＇ s jelly of hu- man umbilical cord via multi-enzyme digestion. Flow cytometry was used to test the hUC-MSCs surface markers. Differ- entiation ability of hUC-MSCs was tested. β-galactosidase was used to test senescent associated activity of hUC-MSCs. Methylation specific PCR （MSP） was used to test methylation status of targeted genes during in vitro culture of hUC- MSCs. Western blot was used to test MGMT protein expression. Results （3 - 6） x 106 hUC-MSCs could be isolated from 30 cm-long umbilical cords, hUC-MSCs expressed high levels of CD29, CD73, CD90, CD105, CD44 and low lev- els of CD45, CD34, HLA-DR. hUC-MSCs had the ability to differentiate to adipocytes, osteogentic and chondrocytes. During the late phase of in vitro culture, hUC-MSCs became senescent and MGMT protein was absent due to DNA meth- ylation. Conclusion hUC-MSCs have biological characteristics of mesenchymal stem cell. During in vitro culture, MG- MT appeared methylation with cell passaging. More studies need to be done to explore the relativity of MGMT methyla- tion and cell senescence.

关 键 词：脐带间充质干细胞 体外培养 DNA甲基化 

分 类 号：Q813[生物学—生物工程]