Properties of human bone marrow mesenchymal stem cells in the early phase of adipogenic differentiation in different culture systems

doi:10.3969/j.issn.2095-4344.2016.23.003

Abstract

Abstract:

BACKGROUND: There are various methods to induce adipogenic differentiation of bone marrow mesenchymal stem cells, and the main component for adipogenic induction is indomethacin or rosiglitazone. However, there is a lack of comparative study on the induction efficiency and mechanism among these methods.
OBJECTIVE: To compare the adipogenic responses of human bone marrow mesenchymal stem cells to different induction methods, and to analyze the mechanism underlying different induction efficiency.
METHODS: After isolation and purification, the adipogenic abilities of human bone marrow mesenchymal stem cells in three different culture systems were compared by oil red O staining and lipogenic gene assay. At 0, 1, 3 and 7 days of adipogenensis, mRNA expressions of PPARγ, C/EBPα, Adiponectin and Leptin were detected. At 7 days of adipogenensis, protein expressions of PPARγ and C/EBPβ were detected by western blot assay, and effects of DIMI versus DIMR on phosphorylation of PPARγ at Ser273 were compared.
RESULTS AND CONCLUSION: Findings from oil red O staining and real-time PCR showed that DIMR significantly induced adipogenic differentiation of bone marrow mesenchymal stem cells compared with DIM and DIMI at 7 days of induction. Western blot showed that the protein expressions of PPARγ and C/EBPβ in the DIMI group were significantly higher than those in the DIMR and DIM at 7days of induction. In addition, the ratio of PPARγ phosphorylation at Ser273 was lower in the DIMR group than the DIMI group. To conclude, DIMR has the most potential to induce early adipogenesis of human bone marrow mesenchymal stem cells by weakening the phosphorylation of PPARγ-Ser273.

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, Adipogenesis, Indomethacin, PPAR gamma, Tissue Engineering

CLC Number:

R394.2

Tian Jian-jian, Chi Ying, Song Bao-quan, Du Wen-jing, Han Zhi-bo, Han Zhong-chao. Properties of human bone marrow mesenchymal stem cells in the early phase of adipogenic differentiation in different culture systems[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(23): 3366-3373.

Figures/Tables 2

2.1 骨髓间充质干细胞的鉴定经密度梯度离心法分离的人骨髓间充质干细胞贴壁培养14-20 d可达70%-80%融合，呈平行排列或漩涡样生长(图1A)；成脂诱导14 d后，经油红O染色可见被染成的红色脂滴(图1B)；成骨分化21 d后，经Von Kossa染色可见明显的黑色钙盐沉积(图1C)；采用流式细胞仪检测骨髓间充质干细胞表面标志物显示CD44、CD73、CD90和CD105呈阳性表达，CD14、CD19、CD34、CD45呈阴性表达(图1D)。 2.2 DIMR对骨髓间充质干细胞的成脂诱导作用明显采用DIM，DIMI与DIMR成脂诱导方法对骨髓间充质干细胞诱导6-8 d，通过油红O染色发现，DIMR组细胞内较大脂肪滴形成，成脂程度明显高于DIM和DIMI组(图2)。 Real-time PCR检测不同时间点成脂相关基因PPARγ，adiponectin，LPL，C/EBPα，leptin及FABP4 mRNA表达，结果发现在成脂诱导早期，adiponectin，LPL mRNA表达不断增加，PPARγ、C/EBPα、leptin、FABP4 mRNA在DIMR组中3 d时表达最高(图3)。在成脂诱导第7天时，adiponectin表达在DIM组中基本不变，DIMI组和DIMR组表达明显增加，且DIMR组高于DIMI组。PPARγ和LPL mRNA在DIM、DIMI和DIMR组均明显升高，但在DIMR组增高程度高于其他两组。C/EBPα和FABP4 mRNA在诱导第7天时，DIM组基本无变化，DIMI组和DIMR组均明显增加，但两组差异不明显。Leptin mRNA在DIM、DIMI和DIMR组中均显著增高。DIMR组在成脂诱导第1天和第3天时高于其余两组，但在第7天时呈明显下降趋势。 2.3 不同方法成脂诱导后骨髓间充质干细胞成脂相关蛋白的表达 Western blot检测骨髓间充质干细胞成脂诱导分化第1，3，7天时各组PPARγ和C/EBPβ蛋白表达量。结果显示在成脂诱导第1天时，DIMI和DIMR组PPARγ表达量明显增加，DIM组无明显改变，在第3，7天时各组PPARγ蛋白表达量显著增加(图4)，差异有显著性意义(P < 0.05)。在各时间点，DIMI组均诱导表达更多的PPARγ。DIM，DIMI，DIMR组在成脂诱导第7天时，PPARγ表达量分别增加了2倍，2.8倍，2.2倍。在第7天，DIMI组与DIMR组PPARγ蛋白表达差异有显著性意义(P < 0.05)，这一结果与基因诱导表达情况不完全一致。C/EBPβ蛋白量在第3天时明显增加，第3天与第7天表达量无明显差异，但DIMI组在第3，7天时的诱导表达量显著高于其他两组，差异有显著性意义(P < 0.05)。 2.4 DIMI，DIMR对PPARγ-Ser273位点磷酸化的作用虽然油红O染色及成脂相关基因mRNA均显示DIMR组较DIMI组有更强的诱导成脂效果，但PPARγ及C/EBPβ蛋白水平检测DIMI组对其诱导表达作用却强于DIMR组，考虑到罗格列酮作为PPARγ的强效激动剂，可直接激活PPARγ发挥作用，因而实验继续检测了DIMI、DIMR组PPARγ-Ser273的磷酸化水平，结果显示，诱导分化第7天时两组中pPPARγ-Ser273水平相似，但DIMR组pPPARγ/PPARγ比例明显低于DIMI组(图5)。"

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