Epithelial transdifferentiation of bone marrow mesenchymal stem cells promoted by ascorbic acid

doi:10.3969/j.issn.2095-4344.2017.01.003

Abstract

Abstract:

BACKGROUND: Ascorbic acid has been shown to promote blood vessel elasticity, strengthen bones, and exert an anti-oxidative role.
OBJECTIVE: To study the effect of ascorbic acid on the epithelial transdifferentiation of bone marrow mesenchymal stem cells in vitro by cell and molecular biology experiments.
METHODS: Different concentrations (0, 10, 20, 40 μmol/L) of ascorbic acid were used to deal with bone marrow mesenchymal stem cells. Under light microscope, we observe the morphological changes of bone marrow mesenchymal stem cells, and screened the optimal concentration of ascorbic acid (40 μmol/L) and suitable interventional time (48 hours). Further detection of E-cadherin, N-cadherin, Snail1 and transforming growth factor-β by real-time PCR was conducted. Western blot assay was also used to detect Oct4, Nanog and
Sox2 in bone marrow mesenchymal stem cells.
RESULTS AND CONCLUSION: Under the light microscope, the long-fusiform cells began to differentiate into rounded epithelial cells after 24 hours of ascorbic acid intervention. And this change became more obvious with the increasing of concentrations. After 48 hours, approximately 90% of cells changed as described above. Ascorbic acid upregulated N-, E- cadherin and downregulated Snail1 and transforming growth factor-β, thereby promoting the epithelial transition of bone marrow mesenchymal stem cells. Meanwhile, the expression of Sox2, Nanog, and Oct4, however, did not change significantly.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Ascorbic Acid, Bone Marrow, Mesenchymal Stem Cells, Epithelium, Tissue Engineering

CLC Number:

R394.2

Han Xiao-lin, Li Jing-dong, Wang Wan-ling, Yang Cui, Li Zhi-ying. Epithelial transdifferentiation of bone marrow mesenchymal stem cells promoted by ascorbic acid[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(1): 13-17.

Figures/Tables 2

2.1 骨髓间充质干细胞的分离与鉴定原代培养首次换液后，贴壁细胞多为单个长梭形细胞，偶尔可见几个细胞的克隆，继续培养可见细胞形态均一，呈长梭形，融合时呈漩涡状生长(图1A)。流式细胞仪鉴定干细胞相关细胞表面标记的表达，结果显示，所分离募集原代细胞内高表达干细胞相关分子CD29和CD90，而CD45呈阴性表达(图1B)。 2.2 抗坏血酸处理骨髓间充质干细胞形态变化根据前期抗坏血酸在诱导多能干细胞中应用的研究，选择10，20，40 μmol/L 3个浓度的抗坏血酸处理骨髓间充质干细胞。抗坏血酸处理24 h，光镜下观察骨髓间充质干细胞的形态开始由长梭形逐渐向形态较圆润的上皮细胞转化。随着抗坏血酸浓度的上调，形态学变化越显著。抗坏血酸处理48 h，光镜下约有90%以上的细胞出现以上变化(图2)。 2.3 抗坏血酸上调骨髓间充质干细胞上皮相关分子并下调间质相关分子mRNA表达以40 μmol/L抗坏血酸处理48 h，采用分子生物学手段观察骨髓间充质干细胞上皮及间质相关分子的mRNA表达。结果显示，上皮相关分子E-钙黏蛋白及N-钙黏蛋白的mRNA表达分别上调(3.3±0.2)倍和(4.1±0.9)倍，P均< 0.001；而间质相关分子snail1及转化生长因子β的mRNA表达分别下调(2.4±0.7)倍和(1.5± 0.4)倍，P均< 0.001，见图3。以上研究说明抗坏血酸可通过上调骨髓间充质干细胞上皮相关分子并下调间质相关分子的mRNA表达，促进骨髓间充质干细胞间质上皮转化。 2.4 抗坏血酸在促进骨髓间充质干细胞间质上皮转化的同时并维持干细胞干性 40 μmol/L抗坏血酸处理48 h后骨髓间充质干细胞内Oct4，Sox2，Nanog蛋白表达均上调，分别为(0.3±0.2)倍，(0.1±0.9)倍，(0.80±0.09)倍，与对照组比较，差异无显著性意义(P > 0.05，图4)。以上结果显示，抗坏血酸在促进骨髓间充质干细胞间质上皮转化的同时，骨髓间充质干细胞干性未发生明显变化，提示抗坏血酸促进骨髓间充质干细胞上皮转分化。"

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