Immortalization and screening of cardiac progenitor cells in mice

doi:10.3969/j.issn.2095-4344.2014.23.018

Abstract

Abstract:

BACKGROUND: Transplantation of exogenous stem cells for functional cardiac cell death or apoptosis easily induced complications after transplantation. Therefore, cardiac progenitor cells of heart itself have been ideal seed cells.

OBJECTIVE: To establish stable cell lines of cardiac progenitor cells from mouse heart, and to provide ideal cell models for studying proliferation and differentiation factors affecting cardiac progenitor cells during adult myocardial cells were damaged.

METHODS: (1) Myocardiocytes were isolated from embryonic 15.5 days mice. (2) The cultured myocardiocytes were immortalized using retrovirus SSR69. Immortalized monoclonal myocardial cells were obtained using antibiotic selection and infinite dilution. (3) Monoclonal cell line with the property of cardiac progenitor cells was screened out.

RESULTS AND CONCLUSION: (1) Myocardiocytes were successfully isolated and cultured. Partial cells showed obvious beating. (2) Myocardiocytes infected with retrovirus SSR69 were cloned and got 76 clones, then were named as CP15-#, (3) Screening the first 20 clones, the reasonable clones with the characteristics of cardiac progenitor cells were obtained according to myocardial cell marker genes. The results suggested that immortalized cardiac progenitor cells were established mediated by reversible SV40 T antigen.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: myocytes, cardiac, clone cells, genes

CLC Number:

R394.2

Zhu Gao-hui, He Tong-chuan, Xiong Feng, Zhu Min1, Chen Yuan. Immortalization and screening of cardiac progenitor cells in mice[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(23): 3715-3722.

Figures/Tables 6

2.1 小鼠心肌特异性基因在心脏发育过程中的表达时序半定量RT-PCR检测胚胎13.5 d(E13.5)、胚胎18.5 d (E18.5)，出生后1 d(D1)、3 d(D3)、7 d(D7)、14 d(D14)和8周的成年小鼠心肌细胞发育基因的表达时序。由图2可见，胰岛因子1特异地表达在心肌细胞E13.5的早期阶段，E18.5时已经几乎检测不到；c-kit在E13.5到成体心脏的发育过程中，表达量逐渐降低，心肌谱系早期表达基因Nkx2.5随心脏的发育成熟而逐渐降低，晚期结构蛋白α-肌球蛋白重链、α-肌动蛋白和心肌蛋白T则随心脏发育成熟而逐渐增加。 2.2 永生化心肌细胞的单克隆生长取孕15.5 d的CD1小鼠胚胎心肌细胞培养，经永生化及潮霉素筛选后，应用无限稀释法获取单克隆，接种后3 d可见单个细胞的生长，第5天时基本可见到细胞分裂形成两三个细胞，第7天时则出现了一个小的细胞克隆团，第10天时细胞克隆团基本达到1/3孔面积(图3)。 2.3 半定量RT-PCR筛选心肌祖细胞单克隆根据图2检测心肌细胞相关基因表达时序的结果，结合文献报道[9-13]，选取不同阶段的心肌细胞标志基因，通过RT-PCR筛选永生化的心肌祖细胞。选用基因八聚体结合转录因子3/4(Oct3/4)、Nanog、Sry相关的HMG盒-2(Sox2)作为干细胞的标志基因，由图4可以看出，20个克隆中基本上都表达这些基因(图4A)。心肌祖细胞标志胰岛因子1在CP15-5A，CP15-9，CP15-10，CP15-13，CP15-18中有表达。干细胞抗原1和c-kit这两个非特异性祖细胞标志也在大多数克隆中有表达(图4B)。心肌谱系早期标志Nkx2.5、GATA结合因子4、Tbox-5、心脏神经嵴衍生表达蛋白2(Hand2)和胎肝激酶-1在CP15-5A，CP15-9， CP15-10，CP15-13，CP15-18均有不同程度的表达，其中Tbox-5表达量较高，且差异比较明显(图4C)。心肌细胞结构蛋白标志α-肌球蛋白重链、α-肌动蛋白、心肌蛋白T和心房利钠因子(ANF)表达量相对较低，且无明显差异(图4D)。将这些电泳图在BIO-RAD电泳仪上经quantity1软件分析后生成图表，转为数值比较。 2.4 免疫荧光进一步检测胰岛因子1和c-kit在胰岛因子1-Tbox-5阳性克隆中表达情况为了进一步验证RT-PCR结果，实验应用免疫光法检测胰岛因子1和c-kit在胰岛因子1-Tbox-5阳性克隆中表达情况，结果显示同RT-PCR检测结果一致(图5)。 2.5 永生化细胞的可复性和永生化基因对细胞分化影响 AdCre感染永生化的细胞CP15-9和CP15-10后，Western Blot检测anti- SV40T表达，2组均可看出Cre可以有效去除SV40T在心肌祖细胞克隆中的表达(图6)。永生化基因SV40T在细胞中的存在是否对细胞增殖有很大影响呢？在永生化心肌祖细胞中分别加入合适滴度的Adcre后，进行细胞计数，加入AdCre组细胞增殖相对减慢，但总体看来AdCre和AdRFP组细胞生长趋势一致，这与预期结果一致(图7)。但是，永生化基因SV40T在细胞中的存在是否对细胞分化有很大影响呢？并且为了后续的应用简单，在细胞转染质粒MyHC-GLuc后，加入合适滴度的Adcre或AdRFP，然后用0.5 µmol/L地塞米松诱导分化，激活质粒活性，检测SV40T对分化过程的影响。加入AdCre组细胞增殖相对减慢，因此在MyHC-GLuc活性检测时，为了结果的可比性，实验应用细胞裂解总蛋白量来标准化GLuc读数，均衡细胞量。结果可以看出，相对于空白对照组，地塞米松可以有效诱导MyHC-GLuc的活性表达(P < 0.05)，而且可以看出空白对照组或地塞米松诱导组中AdCre对MyHC-GLuc的活性影响不大(图8)。"

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