摘要
背景:人脐带间充质干细胞提取方法主要有酶消化法和组织块贴壁法,明确两种方法提取的细胞在二维、三维培养下的生物学特性,对组织工程中种子细胞提取方法及培养方式的选择具有重要意义。目的:比较酶消化法和组织块贴壁法提取的人脐带间充质干细胞在二维、三维培养下的生物学特性。方法:酶消化法和组织块贴壁法分离人脐带间充质干细胞,扩增培养至第3代进行二维、三维培养;扫描电镜观察细胞球表征;死活染色观察细胞球存活情况;Alamar Blue检测细胞增殖能力;流式细胞术检测免疫表型CD34、CD44、CD45、CD90、CD105的表达;免疫荧光、免疫印迹检测多能性转录因子Sox2、Nanog、Oct4表达。结果与结论:(1)酶消化法比组织块贴壁法获取细胞时间更短,二维培养下组织块贴壁法的细胞增殖能力优于酶消化法,但在三维培养条件下细胞增殖缓慢且无差异。三维培养3 d后细胞成紧密球体,存活良好。(2)两种方法提取的细胞在二维、三维培养条件下均表达CD44、CD90、CD105,不表达CD34、CD45。二维培养下,酶消化法提取细胞的CD105表达高于组织块贴壁法;三维培养下,酶消化法提取细胞的CD90、CD105表达也高于组织块贴壁法。(3)二维和三维培养下两种方法提取细胞均表达多能性转录因子Sox2、Nanog、Oct4。二维培养下,酶消化法提取细胞3个转录因子表达均高于组织块贴壁法;但三维培养下,组织块贴壁法提取细胞Sox2的表达高于酶消化法,Nanog、Oct4的表达两组间无差异。
BACKGROUND:The human umbilical cord mesenchymal stem cells isolation methods mainly include enzymatic digestion and tissue attachment.It is important to understand the biological characteristics of the two methods isolated cells in two-dimensional and three-dimensional culture conditions;it is of great significance to the selection of cell isolation methods and culture condition in tissue engineering.OBJECTIVE:To compare the biological characteristics of human umbilical cord mesenchymal stem cells from the enzymatic digestion and tissue attachment methods in two-dimensional and three-dimensional culture conditions.METHODS:Human umbilical cord mesenchymal stem cells were isolated by digestion method and tissue attachment method,amplified and cultured to passage 3 and divided into two-dimensional and three-dimensional culture.Scanning electron microscope was used to observe the cell spheroids.Live\dead staining was utilized to detect cell spheroid survival.Alamar Blue was applied to detect cell proliferation.Flow cytometry was used to detect the expression of immunophenotypes CD34,CD44,CD45,CD90,and CD105.Immunofluorescence and western blot assay were used to detect the expression of pluripotent transcription factors Sox2,Nanog,and Oct4.RESULTS AND CONCLUSION:(1)Enzymatic digestion method took shorter time to obtain cells than the attachment method.The cell proliferation ability of the attachment method was better than that of the enzyme digestion under two-dimensional culture,but the proliferation was slow and there was no difference under three-dimensional culture conditions.After 3 days of spheroid culture,the cells became compact spheroids and survived well.(2)The cells isolated by the two methods all expressed CD44,CD90,and CD105 under two-dimensional and three-dimensional culture conditions,but did not express CD34 and CD45.Under two-dimensional culture,the expression of CD105 cells isolated by enzymatic digestion was higher than that of tissue attachment method;under threedimensional culture,the expression of CD90 and CD105 cells isolated by enzymatic digestion was also higher than that of tissue attachment method.(3)Both isolation methods under two-dimensional and three-dimensional culture expressed pluripotency transcription factors Sox2,Nanog,and Oct4.Under the twodimensional culture,the expression of the three transcription factors of the cells extracted by the enzymatic digestion method was higher than that of the tissue attachment method.However,under the three-dimensional culture,the expression of Sox2 in the tissue attachment method was higher than that of the enzymatic digestion method,and the expression levels of Nanog and Oct4 were not different between the two groups.
作者
陈思铭
胡加伟
李丽丽
伍永强
彭维杰
Chen Siming;Hu Jiawei;Li Lili;Wu Yongqiang;Peng Weijie(Gannan Medical University,Ganzhou 341000,Jiangxi Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2022年第19期2997-3003,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81860327),项目负责人:彭维杰。
关键词
干细胞
脐带
间充质干细胞
三维培养
细胞球
生物学特性
免疫表型
转录因子
stem cells
umbilical cord
mesenchymal stem cells
three-dimensional culture
cell pellets
biological characteristics
immunophenotype
transcription factors
作者简介
第一作者:陈思铭,男,1996年生,江西省赣州市人,汉族,赣南医学院在读硕士,主要从事干细胞、组织工程、生物打印研究。https://orcid.org/0000-0002-8904-1903;通讯作者:彭维杰,博士,教授,硕士、博士研究生导师,赣南医学院,江西省赣州市341000。
