Chinese Journal of Tissue Engineering Research ›› 2014, Vol. 18 ›› Issue (19): 2968-2974.doi: 10.3969/j.issn.2095-4344.2014.19.003
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Zhang Liang-ming1, Wang Xuan1, Gao Wen-jie2, Huang Dong-sheng2, Rong Li-min1
Revised:
2014-03-07
Online:
2014-05-07
Published:
2014-05-07
Contact:
Rong Li-min, M.D., Professor, Chief physician, Doctoral supervisor, Department of Spine Surgery, the Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China
About author:
Zhang Liang-ming, M.D., Physician, Department of Spine Surgery, the Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China
Supported by:
the National Natural Science Foundation of China, No. 81301524, 81171674; the Natural Science Foundation of Guangdong Province, No. S2013040015484; the Specialized Research Fund for the Doctoral Program of Higher Education, No. 20110171110067
CLC Number:
Zhang Liang-ming, Wang Xuan, Gao Wen-jie, Huang Dong-sheng, Rong Li-min . Melatonin inhibits adipogenic differentiation of bone marrow mesenchymal stem cells through retinoid-related orphan receptor alpha[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(19): 2968-2974.
2.1 原代骨髓间充质干细胞的形态与鉴定 骨髓稀释液经过密度梯度离心后,骨髓稀释液被分成数层:血清、单个核细胞层、淋巴细胞分离液、血细胞层。小心吸取呈云雾状单个核细胞层进行原代细胞培养。培养早期以悬浮、折光性强的圆形细胞为主,通过换液悬浮细胞逐渐被清除。骨髓间充质干细胞开始时呈散在细胞克隆生长,为梭形纤维状,数量为3-10个不等。细胞克隆逐渐铺长,最后多个克隆细胞融合在一起,形成梭形纤维状细胞单层,细胞排列有方向性,呈平行、漩涡状生长。一般在10-12 d,细胞可以达到80%-90%融合(图1)。体外培养的第3代骨髓间充质干细胞,采用流式细胞仪计算荧光表达率,检测骨髓间充质干细胞的细胞表面CD分子表达情况。造血干细胞表面标记:CD34表达率2.12%,CD45表达率0.27%;间充质干细胞细胞表面标记:CD29表达率94.81%,CD44表达率94.50%,CD105表达率96.36%(图2)。"
2.2 视黄酸相关孤儿受体α在骨髓间充质干细胞以及其成脂肪分化过程中的表达特征 荧光定量PCR发现骨髓间充质干细胞高表达视黄酸相关孤儿受体α核受体。视黄酸相关孤儿受体α的4种转录异构体中,只有视黄酸相关孤儿受体α1和视黄酸相关孤儿受体α4在骨髓间充质干细胞上表达,其中以视黄酸相关孤儿受体α4表达最为丰富,其表达量约为视黄酸相关孤儿受体α1的2倍(图3)。视黄酸相关孤儿受体α2和视黄酸相关孤儿受体α3在骨髓间充质干细胞没有表达。 在不同浓度的褪黑素的作用下,骨髓间充质干细胞表达视黄酸相关孤儿受体α的量被上调,而且这种作用随着褪黑素的浓度上升而增强(图4A)。进一步,用Western blot检测视黄酸相关孤儿受体α蛋白水平表达量也证实了褪黑素的这种浓度依赖性作用(图4B)。 在骨髓间充质干细胞成脂肪分化过程中,视黄酸相关孤儿受体α在早期(1,3,6 d)有明显的上调表达(P < 0.05),在中后期就维持在相对低水平(图5)。褪黑素抑制成脂肪分化的作用可能与此相关。"
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