Roles of Rev-erb alphya and Ror alpha in osteoblastogenesis of mouse bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.0484

Abstract

Abstract:

BACKGROUND: The orphan nuclear receptors Rev-erbα and Rorα play important roles in lipid metabolism and glucose metabolism. But it is unclear whether they are involved in bone metabolism.
OBJECTIVE: To observe the expression of Rev-erbα and Rorα during the osteoblastogenesis process of bone marrow mesenchymal stem cells (BMSCs).
METHODS: BMSCs were isolated and purified from C57BL/6 mice by the whole bone marrow adherence method followed by morphology observations and then BMSCs were induced to differentiate into osteoblasts and adipocytes. We detected their differentiation abilities using oil red O staining and alizarin red staining, respectively. Real-time PCR and western blot assay were conducted to detect the mRNA and protein levels of Rev-erbα and Rorα in BMSCs cultured in the osteogenic medium for 0, 7, 14 days.
RESULTS AND CONCLUSION: BMSCs from C57BL/6 mice were mainly spindle-shaped and exhibited the swirl-like pattern of growth. Following induction, oil red O staining and alizarin red staining produced a positive reaction in these cells. Rev-erbα expression at both gene and protein levels decreased at 0, 7, 14 days after osteogenic induction, while Rorα expression increased at both gene and protein levels. These findings indicate that Rev-erbα and Rorα may participate in the osteoblastogenesis of BMSCs.

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: Bone Marrow, Mesenchymal Stem Cells, Orphan Nuclear Receptors, Cell Differentiation, Osteoblasts, Tissue Engineering

CLC Number:

R394.2

Lin Fu-wei, Xu Xiao-mei, Cui Yan, Xie Yi-jia, Zhao Qing. Roles of Rev-erb alphya and Ror alpha in osteoblastogenesis of mouse bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(17): 2625-2630.

Figures/Tables 1

2.1 骨髓间充质干细胞形态骨髓细胞刚接种时，悬浮于培养基中呈高亮圆形，大小不一，细胞核不能辨认。24 h后大部分细胞贴壁，形态不一，以小圆形为主，部分细胞贴壁伸展，呈泪滴状。4 d左右培养瓶细胞贴附面可见形态相对均一的折光性较强纺锤形细胞，部分贴壁细胞呈克隆样生长。7 d左右细胞呈现多种形态，细胞呈漩涡状排列，克隆交界处出现重叠、融合，细胞融合达80%。传代接种的骨髓间充质干细胞在24-72 h快速增殖，为细胞增殖高峰期，细胞逐渐呈漩涡状、网状或辐射状排列，胞浆丰富，核大，呈椭圆形，5 d左右即可达到80%融合。传代后的细胞体积均较前变大，形态上更加趋于一致，增殖快，胞核明显，呈放射状向周边生长，即典型的漩涡样生长，见图1。 2.2 骨髓间充质干细胞成骨诱导及茜素红染色鉴定结果经成骨诱导液持续培养后，细胞呈复层生长，由梭形变为立方形，细胞体积增大。细胞外基质分泌增多，部分细胞重叠生长。21 d后茜素红染色可见大量红色矿化结节(图2A)。 2.3 骨髓间充质干细胞成脂诱导及油红O染色鉴定结果经成脂诱导液培养至第7天左右，细胞形态由梭形变为圆形，体积增大，细胞胞浆内含有大量脂滴。至14 d时大量的间充质干细胞转变为脂肪细胞。油红O染色可见胞浆内有较多橙红色折光性强的脂滴(图2B)。 2.4 骨髓间充质干细胞成骨诱导后Rev-erbα及Rorα表达的变化在骨髓间充质干细胞成骨诱导的第0，7，14天分别行Real Time PCR检测Rev-erbα及Rorα基因mRNA水平，Western Blot检测Rev-erbα及Rorα蛋白表达水平。 PCR结果显示：Rev-erbα基因mRNA水平呈下降趋势，见图3A。Rorα基因mRNA表达水平呈上升趋势，见图3B。 Western Blot结果显示：Rev-erbα蛋白表达呈下降趋势，见图3C。而Rorα蛋白表达呈上升趋势，见图3D。"

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