人脐带间充质干细胞体外培养过程中的表观遗传学改变

发布时间：2018-04-24 23:13

本文选题：脐带间充质干细胞 + 体外培养　；参考：《胃肠病学和肝病学杂志》2012年06期

【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。
[Abstract]:Objective to isolate and culture human umbilical cord mesenchymal stem cells (hUC-MSCsS) and to investigate the epigenetic changes during in vitro culture. Methods Human umbilical cord (HUC-MSCs) was obtained from umbilical cord glia by complex enzyme digestion. Cell surface markers were detected by flow cytometry. Differentiation medium induced hUC-MSCs differentiation. 尾-galactosidase staining was used to detect cell senescence. Methylation specific PCR MSPs were used to detect the methylation status of specific genes in hUC-MSCs at different culture stages. Western blot was used to detect the protein expression of MGMT gene in different culture stages of hUC-MSCs. Results according to the 30 cm of the whole umbilical cord, the harvest of UC-MSCs was 6 脳 10 6 h. CD29, CD73, CD105, CD90 and CD44 were highly expressed in cultured hUC-MSCs, but CD45, CD34 and HLA-DR.hUC-MSCs could be differentiated into lipids, osteoblasts and chondroblasts. With the passage of cells, aging cells increased in proportion to the total number of cells. MGMT gene methylation occurred in hUC-MSCs at the late stage of culture. Conclusion combined enzyme digestion can obtain mesenchymal stem cell (MSCs) cells from umbilical cord glia. The methylation of MGMT may be involved in the regulation of hUC-MSCs senescence with the increase of passage times.
【作者单位】：内蒙古大学生命科学学院;内蒙古干细胞医学工程技术研究中心;北京京蒙高科干细胞技术有限公司;中国人民解放军总医院消化科;
【基金】：内蒙古自然科学基金资助项目(2011BS0507)
【分类号】：R329

【相似文献】