Optimal concentration of prostaglandin E2 for osteogenic differentiation of bone marrow mesenchymal stem cells and its effectors

doi:10.3969/j.issn.2095-4344.2014.14.003

Abstract

Abstract:

BACKGROUND: Prostaglandin E2 is not only involved in the body’s inflammatory response as an inflammatory mediator, but also plays a very important role in the process of bone formation and remodeling. In addition, prostaglandin E2 also regulates the proliferation and differentiation of other stem cells, such as neural stem cells and embryonic stem cells.
OBJECTIVE: To observe the effects of prostaglandin E2 on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells and to confirm the best concentration of prostaglandin E2, then to screen differentially expressed genes by gene chip technology, and to study the mechanism of prostaglandin E2 in the osteoinductive process of bone marrow mesenchymal stem cells.
METHODS: Bone marrow mesenchymal stem cells were isolated and cultured from Sprague-Dawley rats, and prostaglandin E2 was prepared to different concentrations of solutions: 10-2, 10-3, 10-4, 10-5 and 10-6 g/L. Then, passage 3 cells were cultured in prostaglandin E2 solutions, and low-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum. MTT colorimetric assay and quantitative detection of alkaline phosphatase were used to confirm the best concentration of prostaglandin E2 promoting proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells. After 14 days, the gene-chip technique was used to detect the differentially expressed genes.
RESULTS AND CONCLUSION: The experimental data were statistically analyzed, we found that the best concentration of prostaglandin E2 for the proliferation of bone marrow mesenchymal stem cells was 1×10-4 g/L, and the best concentration of prostaglandin E2 for the osteogenic differentiation of bone marrow mesenchymal stem cells was 1×10-5 g/L. According to the gene chip, Cxcl13, Cd55 gene and PPAR, MAPK signaling pathways might be the key genes and pathways in the osteogenic differentiation process of bone marrow mesenchymal stem cells.

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, bone marrow, mesenchymal stem cells, prostaglandins E, electrophoresis, microchip

CLC Number:

R394.2

Li Jie-ruo, Deng Si-yuan, Hou Hui-ge, Huang Hao, Wang Huan, Huan Song-wei, She Guo-rong, Luo Si-min, Liu Ning, Zha Zhen-gang. Optimal concentration of prostaglandin E2 for osteogenic differentiation of bone marrow mesenchymal stem cells and its effectors [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(14): 2147-2152.

Figures/Tables 2

2.1 骨髓间充质干细胞形态观察及鉴定形态观察：骨髓间充质干细胞生长良好，呈旋涡状，见图1A。生长周期：FACS检测细胞周期提示G0/G1期：92.32%，G2期：2.45%，S期：5.23%，见图1B。表面抗原测定：流式细胞仪检测细胞表面抗原：CD29表达为92.01%，CD44表达为90.52%，CD34表达为0.36%，CD45表达为0.78%，见图2，符合骨髓间充质干细胞特征。成骨诱导：P3代骨髓间充质干细胞成骨诱导14，21 d分别作碱性磷酸酶染色及茜素红染色，见图3。 2.2 前列腺素E2促增殖实验采用完全随机的单因素方差分析不同前列腺素E2浓度干预骨髓间充质干细胞增殖后的A值，MTT比色法和碱性磷酸酶试剂盒检测1×10-4 g/L的前列腺素E2组与空白组的比较，差异有显著性意义(P < 0.01)，其他浓度组与空白组之间比较，差异无显著性意义(P > 0.05)，最佳促增殖浓度1×10-4 g/L，见表1。 2.3 前列腺素E2促成骨分化实验采用完全随机的单因素方差分析，比较前列腺素E2不同浓度组干预P3代骨髓间充质干细胞分泌碱性磷酸酶的A值，1×10-5 g/L 前列腺素E2组碱性磷酸酶的A值高于空白组及其他浓度前列腺素E2组(P < 0.05)，其他浓度组与空白组之间比较，差异无显著性意义(P > 0.05)。结果说明前列腺素E2具有促骨髓间充质干细胞成骨分化的作用，其最佳浓度为1×10-5 g/L，见表1。 2.4 前列腺素E2促成骨分化能力评价采用完全随机的单因素方差分析，1×10-5 g/L最佳诱导成骨组、经典成骨诱导组及空白A值分别为0.180 35±0.008、0.204 63±0.011 6、0.180 35±0.008 4，3组之间比较，碱性磷酸酶试剂盒对诱导15 d后骨髓间充质干细胞向成骨细胞分化过程中分泌的碱性磷酸酶进行测定。最佳诱导浓度组和经典组分别与空白组进行比较，差异有显著性意义(P值分别为P=0.000 < 0.01、P=0.000 < 0.01)。 2.5 前列腺素E2促成骨分化中差异表达基因的筛选 Affymetrix公司3’端表达谱(大鼠基因组230 2.0芯片)对前列腺素E2诱导前的骨髓间充质干细胞的cDNA进行检测，结果用Expression Console Version 1.1 图像分析软件对芯片图像进行分析，把图像信号转化为数字信号，以2倍差异显著性为标准，Ratio值≥2为上调基因，Ratio值≤0.5为下调基因。在前列腺素E2组中筛选出差异表达基因68条，其中上调基因21条，下调基因47条。在差异表达基因中找到了一组基因，其中表达上调的有CXCL13、CD55基因等，下调的Cxcl1、Abca4、Gucy1a3、Angptl4、Inpp4b、Id2、Fos、Sema6d基因等(图4)[20-27]。其中这些基因参与调控的与细胞增殖及成骨分化相关的信号通路包括PPAR、MAPK等。"

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