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人胚胎骨髓间充质干细胞的增殖能力及其向三个胚层起源细胞分化的特性

王跃春, 张洹

暨南大学医学院血液病研究所.广东,广州 510632

摘要

为探讨人胚胎骨髓间充质干细胞(mesenchymal stem cells from human fetal bone marrow, hfBM--MSCs)增殖能力及向三个胚层起源细胞分化的“类胚胎干细胞”特性，该文利用细胞差速贴壁生长特性分离纯化hfBM--MSCs，用流式细胞仪检测hfBM--MSCs的细胞周期和MSCs特异的表面标志；用免疫细胞化学方法和RT--PCR法分别在蛋白水平和RNA水平检测人端粒酶逆转录酶(human telomerase reverse transcriptase, hTERT)和胚胎干细胞特异性抗原Oct4、SSEA--4的表达；用各种化学试剂诱导hfBM--MSCs向神经元、脂肪细胞和胰岛B细胞分化并加以鉴定。流式细胞仪检测结果显示，第4、5代hfBM--MSCs分别有92.3%和96.1%的细胞处于G_(0)/G_(1)期；这些细胞表达CD29、CD44和CD106, 不表达造血细胞标志CD34和CD45, 极低地表达与移植物抗宿主病(graft--versus--host disease, GVHD)相关的HLA--DR、CD40和CD80。免疫细胞化学和RT--PCR结果显示，hfBM--MSCs表达Oct4、SSEA--4等胚胎干细胞标志， hTERT也呈阳性表达。在特定诱导条件下，hfBM--MSCs可分化为神经元样细胞(外胚层)、脂肪样细胞(中胚层)和胰岛B细胞(内胚层)。因此，人胚胎骨髓中含有丰富的MSCs，hfBM--MSCs具有胚胎干细胞的主要生物学特性，且免疫原性弱，是组织工程和细胞治疗较为理想的种子细胞。

关键词： 胎儿; 骨髓; 间充质干细胞; 胚胎干细胞

Proliferative capacity of mesenchymal stem cells from human fetal bone marrow and their ability to differentiate into the derivative cell types of three embryonic germ layers

Wang Yuechun, Zhang Yuan

Institute of Hematology,Medical College,Jinan University.Guangzhou 510632,Guangdong

Abstract

Strong proliferative capacity and the ability to differentiate into the derivative cell types of three embryonic germ layers are the two important characteristics of embryonic stem cells. To study whether the mesenchymal stem cells from human fetal bone marrow (hfBM-MSCs) possess these embryonic stem cell-like biological characteristics, hfBM-MSCs were isolated from bone barrows and further purified according to the different adherence of different kinds of cells to the wall of culture flask. The cell cycle of hfBM-MSCs and MSCs-specific surface markers such as CD29, CD44, etc were identified using flow cytometry. The expression of human telomerase reverse transcriptase (hTERT), the embryonic stem cell-specific antigens，such as Oct4 and SSEA-4 were detected with immunocytochemistry at the protein level and also tested by RT-PCR at the RNA level. Then, hfBM-MSCs were induced to differentiate toward neuron-like cells, adipose cells, and islet B cells under certain conditions. It is found that 92.3% passage-4 hfBM-MSCs and 96.1% passage-5 hfBM-MSCs were at G_(0)/G_(1) phase respectively. The hfBM-MSCs expressed CD44, CD106 and adhesion molecules CD29, but not antigens of hematopoietic cells CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR, CD40 and CD80. The hfBM-MSCs expressed the embryonic stem cell-specific antigens such as Oct4, SSEA-4, and also hTERT. Exposure of these cells to various inductive agents resulted in morphological changes towards neuron-like cells, adipose cells, and islet B cells and they were tested to be positive for related characteristic makers. These results suggest that there are plenty of MSCs in human fetal bone marrow, and hfBM-MSCs possess the embryonic stem cell-like biological characteristics, moreover, they have a lower immunogenic nature. Thus, the hfBM-MSCs provide an ideal source for tissue engineering and cellular therapeutics.

Key words: Fetus;Bone marrow;mesenchymal stem cells;embryonic stem cell

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引用本文：

王跃春, 张洹. 人胚胎骨髓间充质干细胞的增殖能力及其向三个胚层起源细胞分化的特性[J]. 生理学报 2008; 60 (3): 425-430.

Wang Yuechun, Zhang Yuan. Proliferative capacity of mesenchymal stem cells from human fetal bone marrow and their ability to differentiate into the derivative cell types of three embryonic germ layers. Acta Physiol Sin 2008; 60 (3): 425-430 (in Chinese with English abstract).