CAJ | 학술논문

目的：探讨肝细胞生长因子（HGF）联合5-氮胞苷（5- aza）体外诱导骨髓间充质干细胞（BMMSCs）向心肌样细胞分化的机制。方法①采用全骨髓贴壁法培养 BMMSCs，用流式细胞仪对阳性表面标记抗原 CD44、CD29和阴性标记抗原 CD34进行检测。②取第4代 BMMSCs 进行分组诱导，空白对照组（普通培养基）、5- aza 组（10μmol/ L）、HGF 组（10 ng/ ml）、5- aza + HGF 组（10μmol/ l +10 ng/ ml）。各组 BMMSCs 诱导4周后，实时荧光定量 PCR 法检测各组α- ac-tin、GATA -4相对表达量，免疫组化法鉴定肌钙蛋白 T（cTnT）表达。结果①大鼠 BMMSCs 体外培养形态特征及鉴定：细胞呈长梭形、同心圆状生长，形态逐渐趋向一致。CD44阳性表达率为（86.14）%，CD29阳性表达率为（98.97）%，CD34阳性表达率为（6.86%）；②BMMSCs 诱导4周后空白对照组细胞死亡，免疫组化显示3组均表达心肌特异性结构蛋白 cT-nT；实时荧光定量 PCR 显示3组均表达心肌分化早期基因，其中5- aza + HGF 组明显高于其他两组。结论①骨髓间充质干细胞经5- aza、HGF 诱导后可以分化为心肌样细胞。②在骨髓间充质干细胞诱导为心肌样细胞的过程中，5- aza +HGF 组优于5- aza 及 HGF 组。
목적：탐토간세포생장인자（HGF）연합5-담포감（5- aza）체외유도골수간충질간세포（BMMSCs）향심기양세포분화적궤제。방법①채용전골수첩벽법배양 BMMSCs，용류식세포의대양성표면표기항원 CD44、CD29화음성표기항원 CD34진행검측。②취제4대 BMMSCs 진행분조유도，공백대조조（보통배양기）、5- aza 조（10μmol/ L）、HGF 조（10 ng/ ml）、5- aza + HGF 조（10μmol/ l +10 ng/ ml）。각조 BMMSCs 유도4주후，실시형광정량 PCR 법검측각조α- ac-tin、GATA -4상대표체량，면역조화법감정기개단백 T（cTnT）표체。결과①대서 BMMSCs 체외배양형태특정급감정：세포정장사형、동심원상생장，형태축점추향일치。CD44양성표체솔위（86.14）%，CD29양성표체솔위（98.97）%，CD34양성표체솔위（6.86%）；②BMMSCs 유도4주후공백대조조세포사망，면역조화현시3조균표체심기특이성결구단백 cT-nT；실시형광정량 PCR 현시3조균표체심기분화조기기인，기중5- aza + HGF 조명현고우기타량조。결론①골수간충질간세포경5- aza、HGF 유도후가이분화위심기양세포。②재골수간충질간세포유도위심기양세포적과정중，5- aza +HGF 조우우5- aza 급 HGF 조。
Objective BMMSCs of rats were separated,cultured and purified in vitro in order to explore the mechanism of directional differ-entiation of BMMSCs into cardiomyocyte - like cells by HGF joint 5 - aza. Methods ①BMMSCs of rats were obtained and cultured with the method of whole bone marrow culture. The positive surface markers as CD44,CD29 and negative marker CD34 of cells were detected by flow cytometry. ②The fourth passaged BMMSCs were divided into 4 groups:cells of control group were untreated;cells of 5 - aza group were treated with 5 - aza at 10μmol/ L for 24 h;cells of HGF group were treated with HGF at 10 ng/ ml for 24 h;and cells of HGF +5 - aza group were treated with HGF at 10 ng/ml and 5 - aza at 10 μmol/ L for 24 h. The expression of cardiomyocyte specific proteins cTnT was detected by immunohistochemical method. The rel-ative expression of GATA -4 and α - actin had been detected by real - time quantitative fluorescence PCR. Results ①Morphological characteristics and identification of rat BMMSCs in vitro:these cells showed elongated spindle in shape. The morphological pattern of cell growth gradually showed as consistent and concentric circles. The detection of BMMSCs showed that 86. 14% of cells expressed surface marker CD44 and 98. 97% of CD29, but only 6. 86% of cells expressed surface marker CD34. ②To induce BMMSCs to differentiate into cardiomyocyte - like cells:at 4 weeks after induc-tion,full BMMSCs in control group died;immunohistochemical examination demonstrated that expression of cTnT and cells developed myotube - like structure in 3 groups. Real - time quantitative fluorescence PCR demonstrated the expression of GATA - 4 and α - actin in 3 groups,but the gene segment expression of HGF +5 - aza group had been significantly increased and it was much higher than other groups. Conclusion ①BMMSCs in 5 - aza,HGF and 5 - aza + HGF groups can be induced to differentiate into cardiomyocyte - like cells. ②In the process for inducing BMMSCs to dif-ferentiate into cardiomyocyte - like cells,it is better in 5 - aza + HGF group than 5 - aza and HGF groups.