A Notch signaling pathway inhibitor affects chondrogenesis of human umbilical cord mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2014.23.001

Abstract

Abstract:

BACKGROUND: Notch singling pathway is very important for cell proliferation and differentiation, but its role is still unknown during chondrogenesis of human umbilical cord mesenchymal stem cells.

OBJECTIVE: To investigate the effect of N-[N-(3,5-difluorophenacetyl-L-alanyl)]-(S)- phenylglycinet-butyl ester (DAPT) on inducing human umbilical cord mesenchymal stem cell differentiation into chondrocytes.

METHODS: Human umbilical cord mesenchymal stem cells were isolated from human umbilical cord, then were induced to differentiate into chondrocytes. There were four experimental groups: non-induced group, high-glucose Dulbecco’s modified Eagle’s medium containing 5% fetal bovine serum and 1% double antibody; induced group, induced medium containing 6.25 mg/L insulin, 6.25 mg/L transferrin, 10 μg/L transforming growth factor beta 1,

0.1 μmol/L dexamethasone, 50 mg/L vitamin C, 5% fetal bovine serum and 1% double antibody; dimethyl sulfoxide group, induced medium containing 0.1% dimethyl sulfoxide; DAPT group, induced medium containing 5 μmol/L DAPT.

RESULTS AND CONCLUSION: After chondrogenic induction, the morphology of human umbilical cord mesenchymal stem cells became polygon and positive for toluidine blue and immunofluorescence staining; the expression of Jag-1, PS-1, Notch-1 and Hes-1 decreased significantly (P < 0.01). After the addition of DAPT, compared with the induced group, the relative gene expression of Jag-1, PS-1 and Hes-1 decreased markedly (P < 0.01), the relative gene expression of Notch-1 decreased obviously as well (P < 0.05), and the contents of proteoglycan and collagen type II proteins decreased significantly (P < 0.01). At the same time, the relative gene expression of proteoglycan decreased obviously (P < 0.05), and the relative gene expression of collagen type II decreased in part. Notch signaling pathway exists in human umbilical cord mesenchymal stem cells, once chondrogenesis begins, the signaling strength will decline rapidly. DAPT may prevent human umbilical cord mesenchymal stem cells from differentiating into chondrocytes by Jag-1-Notch-1-Hes-1 pathway.

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

全文链接：

Key words: stem cells, mesenchymal stem cells, umbilical cord, chondrocytes, receptors, Notch

CLC Number:

R394.2

Zhang Jia-wen, Luo Er-mei, Wu Yan-hui, Fu Shu-ying, Chen Dan-chun, Yu Li, Tang Ming-qiao. A Notch signaling pathway inhibitor affects chondrogenesis of human umbilical cord mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(23): 3609-3615.

Figures/Tables 2

2.3 蛋白聚糖和Ⅱ型胶原蛋白的定量分析阿利新蓝法检测各组人脐带间充质干细胞的蛋白聚糖含量：与单纯诱导组相比，添加5 μmol/LDAPT后人脐带间充质干细胞细胞外基质中的蛋白聚糖含量明显下降 (P < 0.01)，说明DAPT对蛋白聚糖的表达有抑制作用，但诱导组和二甲基亚砜组之间的差异不明显(P > 0.05)，见图4A。羟脯氨酸法检测各组人脐带间充质干细胞的Ⅱ型胶原蛋白含量：与单纯诱导组相比，添加5 μmol/L DAPT后人脐带间充质干细胞的Ⅱ型胶原蛋白含量明显下降(P < 0.01)，说明DAPT对Ⅱ型胶原蛋白的表达有抑制作用，但诱导组和二甲基亚砜组之间的差异不明显(P > 0.05)，见图4B。 2.4 实时定量荧光PCR检测相关基因的表达软骨诱导分化前后Notch信号分子的基因表达：与无诱导组相比，诱导组人脐带间充质干细胞在软骨诱导分化后Jag-1、PS-1、Notch-1和Hes-1的基因表达均明显下降(P < 0.01)，说明Notch信号存在于人脐带间充质干细胞中，当人脐带间充质干细胞向软骨细胞诱导分化后，Notch信号表达下降(图5)。添加DAPT后Notch信号分子的基因表达：与单纯诱导组相比，添加5 μmol/L DAPT后人脐带间充质干细胞中Jag-1、PS-1、Hes-1的基因表达明显下降(P < 0.01)，Notch-1基因表达也显著降低(P < 0.05)，说明了DAPT在人脐带间充质干细胞诱导分化后的信号水平上，进一步减弱了Notch信号(图6)。添加DAPT后蛋白聚糖的基因表达：与单纯诱导组相比，添加5 μmol/L DAPT后蛋白聚糖的基因表达明显下降(P < 0.01)，说明了DAPT抑制蛋白聚糖的基因表达，但诱导组和二甲基亚砜组之间的差异不明显(P > 0.05)，见图7A。添加DAPT后Ⅱ型胶原蛋白的基因表达：与单纯诱导组相比，添加5 μmol/L DAPT后Ⅱ型胶原蛋白的基因表达出现一定程度的下降，但诱导组和二甲基亚砜组之间的差异不明显(P > 0.05)，见图7B。"

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