摘要
目的:研究人心包外脂肪干细胞生物学性状,并探讨向心肌细胞诱导时期的情况。为脂肪干细胞移植治疗心肌梗死提供优良的种子细胞。方法:通过胶原酶消化法,由人心包外脂肪组织获得脂肪干细胞,通过细胞免疫学方法检测人心包外脂肪干细胞细胞表面抗原的表达,并定量分析;利用RT-PCR、real-time PCR法定量检测人心包外脂肪干细胞细胞多能性基因(Pluripotent mRNA)的表达;采用血细胞计数板计数并比较诱导获得的心肌细胞数量;免疫组织化学法检测诱导获得的心肌细胞表达心肌特异性抗α-actin、cTnT,进行表型鉴定。结果:人心包外脂肪干细胞体外培养过程中贴壁生长,通过形态学特征以及脂肪干细胞特异性相关抗原检测证实,得到高纯度人心包外脂肪干细胞。其表面标记物CD44、CD90表达阳性,并在3、4代传代细胞中荧光强度达峰值。多能性基因(Oct-4、Rex1、Nanog)的表达随细胞传代而下降。经心肌诱导培养基体外诱导培养后,分化的细胞大多呈肌细胞样,且在3、4代细胞数量达峰值。细胞免疫化学检测证实心肌特异性抗α-actin、cTnT表达阳性。结论:细胞表面荧光强度及多能性基因(Pluripotent mRNA)表达量的变化可影响脂肪干细胞向心肌细胞的诱导,脂肪干细胞处于传代早期时向心肌诱导的可能性大,因此可选择早期向心肌细胞的诱导,这可能为脂肪干细胞移植治疗心肌梗死奠定基础。
Objective：To explore the biological character of the human extrapericardial adipose-derived stem cells,and discuss the period of induction into myocardium,in the treatment of myocardial infarction.Method：The human extrapericardial adipose-derived stem cells were isolated,purified,and digested in 0.1%collagenase.The expression of cell surface specific antigens were detected by immunofluorescence assay.The changes of the pluripotent mRNA were detected by the way of real-time PCR（RT-PCR）.The quality of cells were counted and compared using the blood count plate.The myocardium special antigen（α-actin,cTnT）expressions were detected by immunocytochemical tests.Result：During the in vitro culture,the extraprecardium adipose-derived stem cells adhered to culture flask wall.They were confirmed with the characteristics of morphology and cell surface specific antigens.High purity of the extraprecardium adipose-derived stem cells were obtained.They were positive for CD44,CD90 and the fluorescence intensity reached the peak at passage 3and 4.The adipose-derived stem cells appeared to contain several pluripotent transcription factors（Oct-4,Rex1,Nanog）,but rapidly lose their expression as the extended culture.After induced and cultured with myocardium induction medium,the differentiated cells were muscle-like cells,and the number of cells reached the peak at passages 3and 4.The expression ofα-actin and cardiac troponin T were positive.Conclusion：Changes in the expression pattern of fluorescence intensity and pluripotent markers of human adipose-derived stem cell can influence the induction into myocardial cells.The cells in early to extend to the possibility of induced myocardium,we can choose the early cell passage.This could lay a foundation for the transplantation of human adipose-derived stem cells to treat myocardial infarction.
出处
《临床心血管病杂志》
CAS
CSCD
北大核心
2014年第11期1000-1005,共6页
Journal of Clinical Cardiology
关键词
脂肪干细胞
多能性基因
诱导
分化
人
adipose-derived stem cells
pluripotent gene
induce
differentiation
human
