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新生大鼠心祖细胞原代分离培养及鉴定
Primary culture and identification of cardiac progenitor cells of neonatal rats
目的 原代分离培养及鉴定新生大鼠心祖细胞(CPCs)。 方法 利用差速贴壁原代分离培养CPCs，形态观察和二乙酸羧基荧光素-琥珀酰亚胺酯（CFDA-SE）示踪法检测细胞增殖。使用Fluo-3/AM 钙离子检测法，检测集落增殖细胞中钙离子浓度变化。免疫荧光技术检测集落增殖细胞的干细胞标记物及心肌细胞标记物的表达。 结果 差速贴壁法获得细胞培养1周左右即观察到集落增殖的细胞，圆/椭圆形或不规则多边形细胞呈铺路石样排列。随培养时间延长，细胞集落增大。培养3周左右，细胞集落中细胞形态发生改变（分化），逐渐出现突起或呈梭形等，集落中出现波动/跳动的心肌细胞。用CFDA SE示踪显示，细胞呈集落增殖特点；Fluo-3/AM 钙离子检测显示，集落增殖细胞中分化细胞的钙离子浓度增高。免疫荧光检测显示，不同的集落增殖细胞具异质性，存在c-kit+/CD34- 、Nanog+ /CD34-和c-kit-/Nanog-细胞集落；增殖的细胞集落存在c-kit和Gata4阳性共表达，随细胞分化出现心肌肌钙蛋白T(cTnT)细胞集落。 结论 差速贴壁法原代分离培养的心祖细胞具有集落增殖和分化为心肌细胞的能力，是研究心祖细胞表面标记物、增殖和调控机制的理想材料。
Objective To primarily culture and identify the cardiac progenitor cells (CPCs) in vitro with morphological observation, immunofluorescence and tracing technology. Methods The CPCs were separated and cultivated with differential adhesion methods. The proliferation of CPCs was traced with two carboxyfluorescein succinimidyl ester(CFDA-SE) tracer, observed with a phase contrast microscope. The calcium ion concentration of CPCs was detected with Fluo-3/AM. The expressions of stem cell markers and cardiomyocyte markers were detected with immunoflurescent staining in colony proliferation cells. Results The CPCs of primary culture in vitro with differential adhesion methods showed colony proliferation, in which the undifferentiated cells appeared as circle, oval or irregular and took the arrangement of cobblestone appearance. The cell colony was bigger and the differentiated cells extended to protrude or showed spindles with the extension of incubation time. About 3 weeks, there were the beating myocardial cells in the colony proliferating cells. The CFDA-SE tracing showed that the fluorescence of colony proliferating cells decreased several times. Fluo-3/AM calcium ion inspection showed that its concentration in cardiomyocyte-like cells of colony proliferating cells was more than that in undifferentiated CPCs. Immunoflurescence staining appeared as heterogeneous populations in colony proliferating cells, including c-kit+/CD34-, Nanog+/CD34-and c-kit+/Gata4+ colony. The colony proliferation cells showed cardiac troponin T(cTnT) positive expression. Conclusion The CPCs isolated in vitro with the differential adhesion methods have the characteristic of colony proliferation and differenciation, which can differentiate into cardiomyocytes. They are the best cells to research the biological characteristic, such as, the surface marker, the proliferation mechanism and regulation mechanism.
［1］Hosoda T. C-kit-positive cardiac stem cells and myocardial regeneration ［J］. Am J Cardiovasc Dis, 2012, 2(1):58-67.
［2］French KM, Davis ME. Isolation and expansion of c-kit-positive cardiac progenitor cells by magnetic cell sorting ［J］. Methods Mol Biol, 2014,1181:39-50.
［3］Bergmann O, Bhardwaj RD, Bemard S, et al. Evidence for cardiomyocyte renewal in humans ［J］. Science, 2009, 324(5923): 98-102.
［4］Messina E, DE Angelis L, Frati G, et al. Isolation and expansion of adult cardiac stem cells from human and murine heart ［J］. Circ Res, 2004, 95(9):911-921.
［5］Zhu BB, Cai XH, Sun YP. Induction effect of hepatocyte growth factor and insulin-like growth factor on cardiac stem cells［J］. Acta Anatomica Sinica, 2014, 45(5): 663-669.（in Chinese）
朱蓓蓓，蔡新华，孙银平. 肝细胞生长因子和类胰岛素生长因子对心肌干细胞的诱导作用［J］. 解剖学报, 2014, 45(5): 663-669.
［6］Beltrami AP, Barlucchi L, Torella D. Adult cardiac stem cells are multipotent and support myocardial regeneration ［J］. Cell, 2003,114(6):763-776.
［7］Bearzi C, Rota M, Hosoda T, et al. Human cardiac stem cells ［J］. Proc Nati Acad Sci, 2007, 104 (35): 14068-14073.
［8］Hierlihy AM, Seale P, Lobe CG, et al. The postnatal heart contains a myocardial stem cell population ［J］. FEBS Lett, 2002, 530(13):239-243.
［9］Smith RR, Barile L, Chao HC, et al. Regenerative potential of cardiosphere-derived cells expanded from percutaneous endomyocardial biopsy specimens ［J］. Circulation, 2007, 115(7): 896-908.
［10］Oyama T, Nagai T, Wada H, et al. Cardiac side population cells have a potential to migrate and differentiate into cardiomyocytes in vitro and vivo ［J］. J Cell Biol, 2007, 176(3): 392-341.
［11］Laugwitz KL, Moretti A, Lam J, et al. Postnatal isll+ cardioblasts enter fully differentiated cardiomyocyte lineages ［J］. Nature, 2005, 433(7026):647-653.
［12］Sanada F, Kim J, Czarna A, et al. C-Kit-positive cardiac stem cells nested in hypoxic niches are activated by stem cell factor reversing the aging myopathy ［J］. Circ Res, 2014, 114(1): 41-55.
［13］Brenner C, Franz WM. Pluripotent-stem-cell-derived epicardial cells: a step toward artificial cardiac tissue ［J］.Cell Stem Cell, 2014, 15(5):533-534.
［14］Anversa P, Kajstura J, Leri A,et al. Life and death of cardiac stem cells; a paradigm shift in cardiac biology［J］. Circulation, 2006, 113(11):1451-1463.
［15］Fuentes T, Kenrans-Jonker M. Endogenous cardiac stem cells for the treatment of heart failure ［J］. Stem Cells Cloning, 2013, 25(6):1-12.
［16］Hosoda T. Clonality of mouse and human cardiomyogenesis in vivo ［J］. Proc Natl Acad Sci USA, 2009, 106 (40):17169-17174.
［17］Jia ZhQ, Zhou ChY. Cardiac stem/progenitor cells and repair of heart injury ［J］. Chinese Journal of Biochemistry and Molecular Biology, 2011,27(10):889-893.(in Chinese)
贾竹青，周春燕. 心脏干/粗细胞与心肌损伤修复［J］. 中国生物化学与分子生物学报，2011，27（10）：889-893.
［18］Tang YL, Shen L, Qian K, et al. A novel two-step procedure to expand cardiac Sca-1+ cells clonally ［J］. Biochem Biophys Res Commun, 2007, 359(4):877-883.
［19］Leite CF, Lopes CS, Alves AC, et al. Endogenous resident c-Kit cardiac stem cells increase in mice with an exercise-induced, physiologically hypertrophied heart ［J］. Stem Cell Re, 2015, 15(1): 151-164.
［20］Georgin-Lavialle S, Lhermitte L, Baude C. Blood CD34- c-Kit+ cell rate correlates with aggressive forms of systemic mastocytosis and behaves like a mast cell precursor［J］. Blood, 2011,118(19):5246-5249.
［21］Abdallah KO, Saleh RM, AlShawarby LA, et al. Detection of CD34/CXCR4+ stem cells in peripheral blood of patients following acute myocardial infarction［J］. Egypt J Immunol, 2014, 21(1):27-37.
［22］António N, Soares A, Fernandes R, et al. Endothelial progenitor cells in diabetic patients with myocardial infarction-can statins improve their function［J］? Eur J Pharmacol, 2014, 741:25-36.
［23］da Cunha JM, da Costa-Neves A, Kerkis I, et al. Pluripotent stem cell transcription factors during human odontogenesis［J］. Cell Tissue Res， 2013, 353(3):435-441.
［24］Qi LJ, Guo ZhK, Li Q, et al. Expresssion of Nanog protein in mouse CD73+ adipose tissue derived mesenchymal stem cells ［J］. Acta Anatomica Sinica, 2012, 43(5):619-623. (in Chinese)
齐立杰，郭志坤，李琼，等. Nanog蛋白在CD73＋小鼠脂肪源间充质干细胞中的表达［J］. 解剖学报，2012,43(5):619-623.
［25］Guo YJ, Bing LJ, Liu ShM, et al. Expression of embryonic stem cell-associated factor Nanog in gliomas and its significance ［J］. Acta Anatomica Sinica, 2013, 44 (1):1-5. (in Chinese)
郭雨霁，邴鲁军，刘尚明, 等. 胚胎干细胞关键因子Nanog在胶质廇中表达及意义［J］. 解剖学报, 2013, 44 (1):1-5.
新乡医学院科研项目培育基金
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