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脂肪干细胞与间充质干细胞体外诱导分化为心肌细胞的差别

张端珍, 盖鲁粤, 刘宏伟

沈阳军区总医院先心病内科.辽宁,沈阳 110016；解放军总医院心内科.北京 100853

摘要

该文旨在探讨脂肪干细胞(adipose--derived stem cells, ASCs)和骨髓间充质干细胞(mesenchymal stem cells, MSCs)在组织含量、体外培养和诱导分化为心肌细胞方面的差别。ASCs从新西兰白兔皮下脂肪组织提取，MSCs从大鼠四肢长骨骨髓提取，体外培养扩增，免疫细胞学方法鉴定。采用细胞集落形成法检测组织中干细胞的含量。将不同代的干细胞用不同浓度的5--氮胞苷诱导，观察其形态变化，免疫细胞化学方法检测诱导后细胞是否转化为心肌细胞。结果显示，体外培养的ASCs呈短梭形，分布均匀，生长迅速，细胞形态单一、稳定。MSCs原代生长非常缓慢，呈簇生长，细胞纯度偏低，容易混杂其它细胞类型，传代细胞容易分化和老化。脂肪组织中ASCs含量显著高于骨髓中MSCs含量，且前者含量受年龄影响小。5--氮胞苷诱导ASCs分化为心肌细胞的有效浓度为6---9 #mu#mol/L，而MSCs在3---15 #mu#mol/L 5--氮胞苷诱导下可见心肌细胞形成。诱导后ASCs分化的心肌细胞呈球形细胞团，MSCs分化的心肌细胞呈条形或棒状，且心肌细胞分化率低于ASCs。幼年动物MSCs的组织含量和心肌细胞分化率均高于老年动物，而ASCs受动物年龄影响较小。结果表明，ASCs在组织含量、细胞纯度、生长速度和心肌细胞分化率等方面均明显优于骨髓MSCs，在心肌细胞再生方面较MSCs具有更大的优势。

关键词： 成体干细胞; 细胞分化; 心肌细胞

Differences between adipose--derived stem cells and mesenchymal stem cells in differentiation into cardiomyocytes

Zhang Duanzhen, Gai Luyue, Liu Hongwei

Department of Congenital Heart Disease,the General Hospital of Shenyang Military Region.Shenyang 110016,Liaoning；China

Abstract

Adipose-derived stem cells (ASCs) are similar to bone marrow mesenchymal stem cells (MSCs) in growth kinetics, antigen expression and multi-lineage differentiation capacity. The present study was designed to investigate the differences between ASCs and MSCs in in vitro culture and differentiation into cardiomyocytes. ASCs were isolated from the fatty tissue of New Zealand white rabbits while MSCs were obtained from rat bone marrow. Both ASCs and MSCs were cultured in Iscove's modified Dulbecco's medium supplemented with 15% fetal bovine serum in the same incubator and treated with various concentrations of 5-azacytidine. A clonogenic assay was used to quantify ASCs in fat tissue and MSCs in bone marrow. The number of ASCs in the fatty tissue was much higher than that of MSCs in the bone marrow quantified by clonogenic assay, and MSCs showed a remarkably slow proliferation rate compared with ASCs, especially at primary passage. ASCs began to attach to the bottom of the culture flask 12 h after seeding. The cells in culture assumed a short spindle shape under a phase-contrast microscope and did not form clusters. The phenotype was maintained through repeated subcultures under nonstimulating conditions. No other cell phenotype was observed. MSCs attached to the culture flask at 24-48 h after seeding and grew in clusters. The cells were fibroblast-like and prone to senescence or differentiation into adipose cells. Both ASCs and MSCs before treatment with 5-azacytidine were stained positively for CD29, CD44 and CD105 but negatively for CD34 and CD45, #alpha#-sarcromeric actin, cardiac-specific troponin T and von Willebrand factor. ASCs differentiated into cardiomyocytes only after treatment with 6-9 #mu#mol/L of 5-azacytidine, while MSCs differentiated into cardiomyocytes with 3-15 #mu#mol/L of 5-azacytidine. After treatment with ideal dose of 5-azacytidine, ASCs began to change their morphology and showed multinucleation within the first week and formed a ball-like appearance thereafter, while MSCs showed multinucleation at the second week and formed a stick-like appearance at 3-4 weeks. The percentage of ASC differentiated into cardiomyocytes after treatment with 5-azacytidine was significantly higher than that of MSCs. The age of animal had no significant influence on the tissue percentage, proliferation and differentiation rate of ASCs. However, the tissue percentage of MSCs in bone marrow decreased with increased age of animal and MSCs from old donor rats exhibited less myogenic cells than those from the young after exposure to 5-azacytidine. These results indicate that ASCs have advantages over MSCs in tissue percentage, homology, growth and differentiation rate, suggesting that ASCs are more suitable for cellular cardiomyoplasty than MSCs.

Key words: adult stem cell;cell differentiation;cardiomyocyte

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引用本文：

张端珍, 盖鲁粤, 刘宏伟. 脂肪干细胞与间充质干细胞体外诱导分化为心肌细胞的差别[J]. 生理学报 2008; 60 (3): 341-347.

Zhang Duanzhen, Gai Luyue, Liu Hongwei. Differences between adipose--derived stem cells and mesenchymal stem cells in differentiation into cardiomyocytes. Acta Physiol Sin 2008; 60 (3): 341-347 (in Chinese with English abstract).