成人脂肪间充质干细胞与外周血内皮祖细胞心肌样分化特性比较的实验研究
发布时间：2018-07-15 14:29
【摘要】：目的：分别探讨成人脂肪间充质干细胞（ADMSCs）和成人外周血内皮祖细胞（EPCs）体外分离、培养的方法；证实ADMSCs和EPCs经5-氮胞苷（5-aza）的诱导可分化为心肌样细胞；初步观察心肌特异性转录因子GATA-4和Nkx2.5在诱导过程中表达的变化，并对两种干细胞的心肌样分化特性进行比较，为干细胞移植治疗冠心病时，选择合适的种子细胞提供初步的基础实验支持。 方法：（1）①ADMSCs的分离与培养：外科手术中取成人皮下脂肪组织，利用胶原酶Ⅰ消化法及细胞贴壁法体外分离培养。取生长状态较好的第3代细胞，进行CD29，CD44，Ⅷ因子和人类白细胞抗原（HLA-DR）等表面抗原的间接免疫荧光鉴定，同时设立磷酸盐缓冲溶液（PBS）的空白对照。②ADMSCs的体外诱导：用5-aza对生长状态较好的第3代细胞进行诱导，并对诱导后的细胞进行心肌特异表面标志物结蛋白（Desmin）和肌钙蛋白T（cTnT）的间接免疫荧光鉴定。（2）①EPCs的分离与培养：无菌条件下采集成人外周静脉血，利用密度梯度离心法及细胞贴壁法体外分离培养。原代细胞培养至7d时，进行荆豆凝集素（FITC-UEA-Ⅰ）和乙酰化低密度脂蛋白（Dil-ac-LDL）免疫双荧光鉴定；同时以PBS为空白对照，进行CD133，CD34，KDR等表面抗原的免疫细胞化学染色鉴定。②EPCs的体外诱导：原代细胞培养至7d时，，用5-aza进行诱导，并对诱导后的细胞进行心肌特异表面标志物Desmin和cTnT的间接免疫荧光鉴定。（3）分别在ADMSCs和EPCs诱导后的第7、14、21d，采用PCR方法检测诱导过程中心肌特异性转录因子GATA-4和Nkx2.5的表达情况，并进行比较。 结果：（1）成人脂肪组织中含有长梭形的间充质干细胞，易于在体外分离培养。免疫荧光鉴定CD29，CD44呈阳性表达，Ⅷ因子和HLA-DR呈阴性表达。5-aza诱导ADMSCs后，细胞形态逐渐发生改变，同时增殖速度显著减慢。诱导7d时，细胞呈较为均一的长梭形；诱导至14d时，细胞排列方向逐渐一致，并有聚集成团的倾向；诱导21d时，细胞体积较前增大，呈球形或不规则形，有的有突起伸出，胞浆不均。免疫荧光鉴定Desmin和cTnT呈阳性表达。（2）成人外周血中含有EPCs，可以在体外分离培养。免疫双荧光鉴定呈阳性表达，免疫细胞化学染色鉴定CD133，CD34，KDR呈阳性表达。5-aza诱导EPCs后，细胞形态发生改变，细胞长度增加，逐渐呈均匀一致的长梭形，同时细胞排列方向也渐趋一致；诱导后期有的细胞呈球形或不规则形，有聚集成团倾向。诱导14d时免疫荧光鉴定Desmin和cTnT呈阳性表达。（3）ADMSCs和EPCs诱导后的PCR产物条带可见GATA-4和Nkx2.5基因表达，并且随着诱导时间的逐渐延长，相对表达量增多；同一诱导时间段比较，ADMSCs诱导后的心肌特异基因相对表达显著高于EPCs组(p0.05)。 结论：（1）5-氮胞苷能将体外培养的脂肪间充质干细胞诱导为心肌样细胞；（2）5-氮胞苷能将外周血内皮祖细胞诱导为心肌样细胞，但成功率较低。（3）与内皮祖细胞比较，脂肪间充质干细胞诱导相对容易，诱导后心肌样细胞分化较多，是干细胞移植治疗冠心病时更为理想的种子细胞。
[Abstract]:Objective: To investigate the isolation and culture of adult adipose mesenchymal stem cells (ADMSCs) and adult peripheral blood endothelial progenitor cells (EPCs) in vitro, and to confirm that ADMSCs and EPCs can be differentiated into myocardial like cells by the induction of 5- azytidine (5-aza), and the changes of the expression of GATA-4 and Nkx2.5 in the induction of cardiac specific transcription factors are observed. The comparison of the myocardial differentiation characteristics of two kinds of stem cells is made to provide preliminary basic experimental support for selecting suitable seed cells for stem cell transplantation in the treatment of coronary heart disease.
Methods: (1) the isolation and culture of ADMSCs: taking adult subcutaneous adipose tissue during surgery, using collagenase I digestion method and cell adherence method to isolate and culture in vitro. Third generation cells with better growth status were selected for indirect immunofluorescence identification of surface antigens such as CD29, CD44, factor VIII and human leukocyte antigen (HLA-DR), and were established at the same time. The blank control of phosphate buffer solution (PBS). (2) the induction of ADMSCs in vitro: the induction of third generation cells with better growth state by 5-aza and indirect immunofluorescence identification of the induced cell specific surface marker protein (Desmin) and troponin T (cTnT). (2) the isolation and culture of EPCs: aseptic conditions The adult peripheral venous blood was collected and cultured in vitro by density gradient centrifugation and cell adherence method. When the primary cells were cultured to 7d, the double fluorescence identification of FITC-UEA- I and acetylated low density lipoprotein (Dil-ac-LDL) was carried out, and the immune cells of CD133, CD34, KDR and other surface antigens were carried out with PBS as blank control. Chemical staining identification. (2) induction of EPCs in vitro: induction of primary cell culture to 7d, induced by 5-aza, and indirect immunofluorescence identification of specific surface markers Desmin and cTnT for the induced cells. (3) ADMSCs and EPCs induced 7,14,21d, respectively, to detect the specific transcriptional cause of the central muscle in the induction process by PCR method. The expressions of GATA-4 and Nkx2.5 are compared and compared.
Results: (1) the adult adipose tissue contains long spindle shaped mesenchymal stem cells, which are easy to be isolated and cultured in vitro. The immunofluorescent identification of CD29, CD44 is positive. The cell morphology changes gradually and the proliferation rate slows markedly at the same time when the negative expression of.5-aza is induced by factor VIII and the negative expression of HLA-DR. At the same time, the cells are more homogeneous in the induction of 7D. Spindle shape, when induced to 14d, the direction of cell arrangement is consistent and there is a tendency to gather together. When inducement of 21d, the cell volume is larger than before, it is spherical or irregular, some protruding and uneven cytoplasm. Immunofluorescence identification of Desmin and cTnT is positive. (2) the adult peripheral blood contains EPCs, and can be isolated and cultured in vitro. Double fluorescence identification showed positive expression. Immunocytochemical staining identified CD133, CD34, KDR positive expression of.5-aza induced EPCs, cell morphology changed, cell length increased, and gradually showed uniform long spindle shape, and the direction of cell arrangement gradually became consistent; some cells in the post induction period were spherical or irregular, and there were aggregation groups tilting. The positive expression of Desmin and cTnT was expressed by immunofluorescence in the induction of 14d. (3) the expression of GATA-4 and Nkx2.5 gene in the PCR product bands induced by ADMSCs and EPCs was visible, and the relative expression increased with the gradual prolongation of the induction time. Compared with the same induction time, the relative expression of the specific gene of myocardium after the induction of ADMSCs was significantly higher than that of the EPCs group. (P0.05).
Conclusion: (1) 5- nitrocytidine can induce adipose mesenchymal stem cells cultured in vitro into cardiomyocyte like cells. (2) 5- azacytidine can induce endothelial progenitor cells of peripheral blood into cardiomyocyte like cells, but the success rate is low. (3) compared with endothelial progenitor cells, the inducement of adipose mesenchymal stem cells is relatively easy, and the differentiation of cardiomyocyte like cells is more than that of endothelial progenitor cells. Cell transplantation is a more ideal seed cell for the treatment of coronary heart disease.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2124382
[Abstract]:Objective: To investigate the isolation and culture of adult adipose mesenchymal stem cells (ADMSCs) and adult peripheral blood endothelial progenitor cells (EPCs) in vitro, and to confirm that ADMSCs and EPCs can be differentiated into myocardial like cells by the induction of 5- azytidine (5-aza), and the changes of the expression of GATA-4 and Nkx2.5 in the induction of cardiac specific transcription factors are observed. The comparison of the myocardial differentiation characteristics of two kinds of stem cells is made to provide preliminary basic experimental support for selecting suitable seed cells for stem cell transplantation in the treatment of coronary heart disease.
Methods: (1) the isolation and culture of ADMSCs: taking adult subcutaneous adipose tissue during surgery, using collagenase I digestion method and cell adherence method to isolate and culture in vitro. Third generation cells with better growth status were selected for indirect immunofluorescence identification of surface antigens such as CD29, CD44, factor VIII and human leukocyte antigen (HLA-DR), and were established at the same time. The blank control of phosphate buffer solution (PBS). (2) the induction of ADMSCs in vitro: the induction of third generation cells with better growth state by 5-aza and indirect immunofluorescence identification of the induced cell specific surface marker protein (Desmin) and troponin T (cTnT). (2) the isolation and culture of EPCs: aseptic conditions The adult peripheral venous blood was collected and cultured in vitro by density gradient centrifugation and cell adherence method. When the primary cells were cultured to 7d, the double fluorescence identification of FITC-UEA- I and acetylated low density lipoprotein (Dil-ac-LDL) was carried out, and the immune cells of CD133, CD34, KDR and other surface antigens were carried out with PBS as blank control. Chemical staining identification. (2) induction of EPCs in vitro: induction of primary cell culture to 7d, induced by 5-aza, and indirect immunofluorescence identification of specific surface markers Desmin and cTnT for the induced cells. (3) ADMSCs and EPCs induced 7,14,21d, respectively, to detect the specific transcriptional cause of the central muscle in the induction process by PCR method. The expressions of GATA-4 and Nkx2.5 are compared and compared.
Results: (1) the adult adipose tissue contains long spindle shaped mesenchymal stem cells, which are easy to be isolated and cultured in vitro. The immunofluorescent identification of CD29, CD44 is positive. The cell morphology changes gradually and the proliferation rate slows markedly at the same time when the negative expression of.5-aza is induced by factor VIII and the negative expression of HLA-DR. At the same time, the cells are more homogeneous in the induction of 7D. Spindle shape, when induced to 14d, the direction of cell arrangement is consistent and there is a tendency to gather together. When inducement of 21d, the cell volume is larger than before, it is spherical or irregular, some protruding and uneven cytoplasm. Immunofluorescence identification of Desmin and cTnT is positive. (2) the adult peripheral blood contains EPCs, and can be isolated and cultured in vitro. Double fluorescence identification showed positive expression. Immunocytochemical staining identified CD133, CD34, KDR positive expression of.5-aza induced EPCs, cell morphology changed, cell length increased, and gradually showed uniform long spindle shape, and the direction of cell arrangement gradually became consistent; some cells in the post induction period were spherical or irregular, and there were aggregation groups tilting. The positive expression of Desmin and cTnT was expressed by immunofluorescence in the induction of 14d. (3) the expression of GATA-4 and Nkx2.5 gene in the PCR product bands induced by ADMSCs and EPCs was visible, and the relative expression increased with the gradual prolongation of the induction time. Compared with the same induction time, the relative expression of the specific gene of myocardium after the induction of ADMSCs was significantly higher than that of the EPCs group. (P0.05).
Conclusion: (1) 5- nitrocytidine can induce adipose mesenchymal stem cells cultured in vitro into cardiomyocyte like cells. (2) 5- azacytidine can induce endothelial progenitor cells of peripheral blood into cardiomyocyte like cells, but the success rate is low. (3) compared with endothelial progenitor cells, the inducement of adipose mesenchymal stem cells is relatively easy, and the differentiation of cardiomyocyte like cells is more than that of endothelial progenitor cells. Cell transplantation is a more ideal seed cell for the treatment of coronary heart disease.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
【参考文献】
相关期刊论文 前6条
1 Bartosz Balana;Cecilia Nicolett;Ihor Zahanich;Eva M Graf;Torsten Christ;Sabine Boxberger;;5-Azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells[J];Cell Research;2006年12期
2 邵诗颖;王红祥;李宾公;邹萍;;人外周血来源内皮祖细胞诱导分化为心肌样细胞的实验研究及机制探讨[J];中国病理生理杂志;2007年10期
3 陈绍良,方五旺,钱钧,叶飞,刘煜昊,单守杰,张俊杰,林松,廖联明,赵春华;Improvement of cardiac function after transplantation of autologous bone marrow mesenchymal stem cells in patients with acute myocardial infarction[J];Chinese Medical Journal;2004年10期
4 ;Transplantation of autologous adipose-derived stem cells ameliorates cardiac function in rabbits with myocardial infarction[J];Chinese Medical Journal;2007年04期
5 李崇剑;高润霖;杨跃进;宋来凤;阮英茆;胡奉环;杨伟宪;陈纪林;乔树宾;秦学文;刘玉清;陈在嘉;;猪心肌缺血再灌注损伤区自体骨髓单个核细胞和外周血内皮祖细胞移植后细胞分化探讨[J];中华心血管病杂志;2007年04期
6 张啸飞;胡大一;丁荣晶;王卉呈;颜流霞;;中国心脑血管疾病死亡现况及流行趋势[J];中华高血压杂志;2012年06期
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