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目的:胎儿心脏正处于组织器官形成和发育时期,其中的细胞数量和增殖能力应该高于成体组织,但是目前关于培养人胎儿心脏来源细胞的方法以及基本生物学特征还鲜见报道。实验拟建立体外培养胎儿心脏来源细胞的方法,探讨心脏来源细胞基本的生物学特征和细胞种类。方法:实验于2005-10/2007-05在南京大学医学院和国家生物医药技术重点实验室完成。①实验材料:水囊引产的12～18周胎儿10例由南京大学医学院附属鼓楼医院提供,实验经产妇及家属同意,并经医院医学伦理委员会批准。②实验方法:取胎龄12～18周水囊引产的胎儿心脏,将消化后获得的细胞培养于添加内皮生长因子、碱性成纤维细胞生长因子、干细胞生长因子、心肌营养素及含10% FBS的DMEM/F12培养基中。③实验评估:应用显微镜观察细胞的形态特征,采用RT-PCR检测培养前后心脏来源细胞干细胞标志基因等基因的表达,流式细胞仪检测培养前及培养19d后细胞的表面标志。结果:①心脏来源的细胞可以生长增殖。培养16d后出现克隆,并有类似生长中心的区域形成,类似生长中心区域内的细胞较小,形态接近圆形,细胞核占细胞比例大,而周围的细胞形状多为梭形核多角形,细胞核所占的比例较小,并且显微镜下可见正在分裂的细胞。②RT-PCR检测显示,胎儿心脏组织分离获得的心肌细胞中表达c-Kit、KDR、GATA-4、Nkx-2.5、MEF-2c,不表达c-Tnl。培养19d后细胞中GATA-4、KDR、MEF-2c表达量降低,不表达c-Kit,Nkx-2.5和c-Tnl。③流式细胞仪检测发现培养19d后细胞群中CD29和CD44高表达,CD45、GATA-4和c-Tnt低表达。结论:建立了一种体外培养胎儿心脏来源细胞的方法,并确定细胞群中有心脏来源的间充质干细胞和心肌祖细胞。
OBJECTIVE: The fetal heart is in the period of formation and development of tissues and organs. The number of cells and the proliferative capacity of the fetus should be higher than that of adult tissues. However, the current methods and basic biological features of human fetal heart-derived cells are rarely reported. Experiment to establish a method of in vitro culture of fetal cardiac cells to explore the basic biological characteristics of cardiac cells and cell types. Methods: The experiment was performed at Nanjing Medical College and National Key Laboratory of Biomedicine Technology from October 2005 to May 2007. ① Experimental Materials: 10 fetuses of 12-18 weeks induced by water sac were provided by Drum Tower Hospital attached to Nanjing University Medical School. The experiment was approved by the mothers and their families and approved by the Hospital Medical Ethics Committee. ② Experimental Methods: Fetal heart of 12 ~ 18 weeks gestational age fetus was induced. The cells obtained after digestion were cultured in the medium supplemented with endothelial growth factor, basic fibroblast growth factor, stem cell growth factor, cardiomyocyte nutrient and 10% FBS DMEM / F12 medium. ③ Experimental evaluation: Morphological characteristics of cells were observed under microscope. RT-PCR was used to detect the expression of cardiac-derived stem cell marker genes before and after culture. The surface markers of cells before and 19 days after culture were detected by flow cytometry. Results: ① heart-derived cells can grow and proliferate. After 16 days of culture, the clones appeared and grew in a similar growth center. The cells in a similar growth center were smaller and in the shape of a circle. The nuclei accounted for a large proportion of cells, while the surrounding cells were mostly fusiform polygons and nuclei The proportion of small, and under the microscope can be seen dividing cells. ② RT-PCR showed that c-Kit, KDR, GATA-4, Nkx-2.5 and MEF-2c were expressed in cardiomyocytes isolated from fetal heart tissue without c-Tnl expression. After cultured for 19 days, the expression of GATA-4, KDR and MEF-2c in cells decreased, but c-Kit, Nkx-2.5 and c-Tnl were not expressed. ③The results of flow cytometry showed that the expression of CD29 and CD44 in the cell population was high after 19 days of culture, while the expressions of CD45, GATA-4 and c-Tnt were low. CONCLUSION: A method of culturing fetal cardiac derived cells was established and cardiac derived mesenchymal stem cells and cardiac progenitor cells were identified in the cell population.