脂肪干细胞与骨髓间充质干细胞心肌样分化特性的比较

Characteristics of human adipose-derived stem cells versus bone marrow mesenchymal stem cells differentiating into cardiomyocytes

目的:骨髓间充质干细胞与脂肪干细胞均具有分化为心肌细胞的潜能。观察脂肪干细胞体外诱导分化为心肌细胞的特性及相关基因表达,比较骨髓间充质干细胞与脂肪干细胞心肌分化能力的差异。方法:实验于2006-08/2007-04在吉林省耳鼻咽喉研究所和教育部吉林大学人兽共患病重点实验室完成。①实验材料:脂肪组织标本来源于本院手术患者,所有患者知情同意,术中收集多余的皮下脂肪。②实验方法:分离培养人脂肪干细胞,用5-氮胞苷诱导传4～6代的脂肪干细胞分化,平行培养第5代骨髓间充质干细胞,诱导条件相同,对照组培养液不添加任何诱导因子。③实验评估:采用免疫荧光技术观察脂肪干细胞和骨髓间充质干细胞表达结蛋白Desmin和肌钙蛋白Troponin T的阳性细胞率。通过即时RT-PCR的方法检测诱导过程中细胞内GATA-4和NKX2.5的mRNA水平。结果:脂肪干细胞经5-氮胞苷诱导后3周,肌性基因标志结蛋白desmin和肌钙蛋白Troponin-T阳性细胞明显增加。与骨髓间充质干细胞组比较,脂肪干细胞表达心肌特异蛋白的阳性细胞率在诱导后第14天明显升高,而在第21天时,两组细胞的阳性表达率均达到60%左右,无明显的统计学差别,未加任何诱导条件的脂肪干细胞对照组培养14d时有2.5%和1.6%的细胞表达Desmin,Troponin-T,在骨髓间充质干细胞对照组未见心肌标记蛋白的阳性表达。即时PCR检测结果发现,诱导组与未经诱导组的脂肪干细胞均表达GATA-4和NKX2.5,骨髓间充质干细胞组在诱导后第7天出现GATA-4和NKX2.5的转录活性。结论:体外培养的脂肪干细胞能够被5-氮胞苷诱导分化为心肌样细胞,并且具有自发分化的能力,与骨髓间充质干细胞相比,脂肪干细胞的早期分化特性更有利于心肌干细胞移植治疗。

AIM. Both bone marrow-derived mesenchymal stem cells （BMSCs） and human adipose-derived stem cells （ADSCs） could differentiate into cardiomyocyte. In this study, the cardiomyocyte differentiation potential and transcription regulation of ADSCs and differentiation ability were compared with BMSCs. METHODS： Between August 2006 and April 2007, the experiment was conducted in the Institute of Otolaryngology, Head and Neck Surgery and Key Laboratory of Zoonosis of Jilin University, Ministry of Education. ①ADSCs were isolated from the subcutaneous adipose tissue of the volunteer patients with the informed consent, and the fourth and sixth generations were induced to differentiate by 5-Azacytidine. A parallel cultured BMSC was induced under the same condition; the cells wthout any induced factor in medium served as control group. ②Immunofluorescence was used to detect the positive expression rate of Troponin T and Desmin in ADSCs and BMSCs. Real time RT-PCR was performed to investigate mRNA level of GATA-4 and NKX2.5. RESULTS： Three weeks after induction of 5-Azacytidine, the positive cells of Desmin and Troponin T were significantly increased. Compared with BMSCs, Desmin and Tropnin T positive cell rates of ADSCs group were significantly increased after induced up to 14 days, however, no change （60%） was observe at 21 day. In ADSCs control group, Desmin （2.5%） and Troponin （1.6%） positive cells were found at 14 day after induced, but no positive expression was observed in BMSCs. RT-PCR assessment showed that both normal and induced ADSCs expressed GATA-4 and NKX2.5. Expression of GATA-4 and NKX2.5 in BMSCs groups appeared on the 7^th day after induced. CONCLUSION： In vitro cultured ADSCs possess the spontaneous differentiation potential into cardiomyocyte. Compared with BMSCs, ADSCs are more available to stem cell transplantation to heart due to its differentiation characteristics at early stage.