Overexpression of heparin-binding epidermal growth factor-like growth factor in Prrx1+ bone marrow mesenchymal stem cells inhibits mouse skeletal development

doi:10.3969/j.issn.2095-4344.1805

Abstract

Abstract:

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) plays an important role in bone development. However, there is no report regarding the effects of HB-EGF derived from bone marrow mesenchymal stem cells on skeletal development.
OBJECTIVE: To study the effects of HB-EGF overexpression in bone marrow mesenchymal stem cells on skeletal development and to preliminarily explore the underlying mechanisms.
METHODS: The mouse model of HB-EGF overexpression in Prrx1⁺ bone marrow mesenchymal stem cells(ROSA26^tm1(HB-EGF); Prrx1-Cre) was constructed using Cre-LoxP system. Bone sections were processed and stained for histological analysis. Mouse bone marrow mesenchymal stem cells were cultured in vitro and their differentiation potential and relevant molecular mechanism were assessed. The experiment was approved by the Experimental Animal Management, Academics, Ethics and Use Committee of Shanghai Jiao Tong University in 2015. The approval number is A2015027.
RESULTS AND CONCLUSION: (1) Prrx1 was expressed in the articular cartilage, growth plate, trabecular bone and periosteum of adult mice. (2) The femurs of ROSA26^tm1(HB-EGF); Prrx1-Cre mice were shortened, the bone mass decreased significantly, and the cartilage morphology and structure were abnormal, all of which indicated osteoarthritic phenotype. (3) Overexpression of HB-EGF inhibited the osteogenic and chondrogenic differentiation of bone marrow mesenchymal stem cells from ROSA26^tm1(HB-EGF); Prrx1-Cre mice in vitro. These results suggest that overexpression of HB-EGF in Prrx1⁺ bone marrow mesenchymal stem cells may influence the skeletal development by inhibiting osteogenic and chondrogenic differentiation.

Key words: Prrx1, heparin-binding epidermal growth factor-like growth factor, bone marrow mesenchymal stem cells, bone, osteoblasts, chondrocytes, osteoarthritis, transgenic model

CLC Number:

Liu Jiajia, Li Ping, Deng Qi, Liu Huijuan. Overexpression of heparin-binding epidermal growth factor-like growth factor in Prrx1⁺ bone marrow mesenchymal stem cells inhibits mouse skeletal development[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(29): 4623-4628.

Figures/Tables 2

2.1 实验动物数量分析所有构建的ROSA26-tdTomato; Prrx1-Cre的小鼠均进入结果分析。 2.2 Prrx1+细胞标记小鼠典型的骨结构实验首先构建了ROSA26-tdTomato; Prrx1-Cre小鼠，通过红色荧光来示踪骨中Prrx1的表达。结果显示，Prrx1在股骨关节头、生长板、骨小梁表面和骨膜等典型的骨结构中均有表达，尤其是关节头和生长板区域，见图1。 2.3 ROSA26tm1(HB-EGF); Prrx1-Cre小鼠骨量减少通过X射线成像获得长骨二维图，发现ROSA26tm1(HB-EGF); Prrx1-Cre小鼠呈现股骨变短，关节头变宽的趋势，见图2A。定量分析结果证实其股骨长度从1.4 cm缩短到0.9 cm，关节头宽度从3.1 mm增加到3.8 mm，其差异有显著性意义，见图2B。微型CT三维重建结果发现过表达组骨体积分数降低了90%，骨小梁数量减少70%，骨小梁厚度减少20%，骨小梁分离距离增加1.5倍，见图2C，表明ROSA26tm1(HB-EGF); Prrx1-Cre小鼠骨基质明显减少。 2.4 ROSA26tm1(HB-EGF); Prrx1-Cre小鼠软骨异常 Safranin O染色发现，对照组关节结构完整，关节头及生长板软骨层结构清晰、排列整齐，而ROSA26tm1(HB-EGF); Prrx1-Cre小鼠胫骨关节头凹陷，有异位软骨积聚，股骨和胫骨关节头软骨层变薄，软骨细胞减少，软骨生长板断裂，见图3A。通过免疫组化染色发现，FSP1在ROSA26tm1(HB-EGF); Prrx1-Cre小鼠关节头表达的区域增加，同时阳性细胞的数量也明显增加，表明关节头处纤维化增加，见图3B。综上，ROSA26tm1(HB-EGF); Prrx1-Cre小鼠关节头及生长板软骨细胞紊乱，有关节炎样表型。 2.5 ROSA26tm1(HB-EGF); Prrx1-Cre小鼠骨髓间充质干细胞分化减弱将骨髓间充质干细胞进行体外培养诱导成骨、软骨分化，碱性磷酸酶染色结果发现过表达组成骨细胞明显减少，同时成骨细胞的标志基因Alp、Runx2、Osx、Ocn、Dmp1的表达都显著下调；阿尔辛蓝染色结果发现成熟的软骨细胞明显减少，软骨细胞的标志基因Col2α1、Col10α1、Sox9、Mmp13的表达也显著下降，见图4。这些结果说明过表达HB-EGF能抑制骨髓间充质干细胞成骨、软骨分化。"

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