超顺磁性氧化铁与荧光染料联合标记骨髓间充质干细胞及生物学特性检测

发布时间：2018-02-26 14:28

本文关键词： 骨髓间充质干细胞超顺磁性氧化铁 DAPI CM-DiI 标记　出处：《山西医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)已经成为组织工程中重要的的种子细胞,在多种疾病的临床治疗中具有广阔的应用前景。而若想深入研究BMSCs的存活、增殖、分化及在体内的迁移、归巢情况,首先需要解决如何高效、安全地标记BMSCs的问题。本文通过观察SPIO、DAPI和CM-DiI对家兔BMSCs的联合标记效果及其对细胞存活、增殖以及分化的影响,为BMSCs的活体示踪研究奠定理论及实验基础。方法：分离培养家兔骨髓间充质干细胞,用SPIO、DAPI和CM-DiI联合标记后,采用普鲁士蓝染色法检测SPIO标记率,荧光显微镜检测荧光标记率,透射电子显微镜观察细胞的超微结构及SPIO在细胞内的分布,台盼蓝拒染法检测细胞活力,MTT法检测细胞增殖力,并使用地塞米松、胰岛素、3-异丁基-1-甲基黄嘌呤、吲哚美辛体外诱导标记细胞向脂肪细胞分化并用透射电子显微镜鉴定诱导后细胞,使用5-氮杂胞苷体外诱导标记细胞向心肌细胞分化并用免疫荧光鉴定诱导后细胞。结果：SPIO、DAPI和CM-DiI在体外对家兔BMSCs的联合标记率几乎达100%；透射电镜下可见电子密度高的SPIO颗粒主要分布于溶酶体、线粒体内部及胞内其他膜性结构上,细胞核内未见分布,且联合标记BMSCs的超微结构显示清晰、形态保存良好；台盼蓝拒染法显示联合标记BMSCs组的细胞活力与未标记BMSCs组相比差异无统计学意义(P0.05)；MTT法检测发现联合标记BMSCs组的细胞增殖力与未标记BMSCs组相比差异无统计学意义(P0.05)；联合标记BMSCs经地塞米松、胰岛素、3-异丁基-1-甲基黄嘌呤、吲哚美辛体外诱导后,透射电镜下可见胞浆内出现脂滴；联合标记BMSCs经5-氮杂胞苷体外诱导后,免疫荧光鉴定心肌特异性肌钙蛋白T(cTnT)阳性。结论：SPIO、DAPI和CM-DiI对家兔骨髓间充质干细胞的联合标记率高,并对其存活、增殖以及分化无影响。
[Abstract]:Objective: bone marrow mesenchymal stem cells (BMSCs) have become important seed cells in tissue engineering and have wide application prospects in clinical treatment of various diseases. In the process of differentiation, migration in vivo and homing, we need to solve the problem of how to label BMSCs efficiently and safely. In this paper, we observed the co-labeling effect of SPION DAPI and CM-DiI on rabbit BMSCs and its effects on cell survival, proliferation and differentiation. It lays a theoretical and experimental foundation for the study of BMSCs in vivo. Methods: rabbit bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured. After labeled with SPIO-DAPI and CM-DiI, the labeling rate of SPIO was detected by Prussian blue staining and the fluorescence labeling rate was detected by fluorescence microscope. Transmission electron microscopy (TEM) was used to observe the ultrastructure of cells and the distribution of SPIO in the cells. The viability of cells was detected by trypan blue exclusion assay. The proliferation of cells was detected by using dexamethasone, insulin 3-isobutyl-1-methylxanthine, dexamethasone, insulin 3-isobutyl-1-methylxanthine. Indomethacin induced the differentiation of labeled cells into adipocytes and identified them by transmission electron microscopy. 5-azacytidine was used to induce the differentiation of labeled cells into cardiomyocytes in vitro and immunofluorescence was used to identify the induced cells. Results the combined labeling rate of BMSCs in rabbit with CM-DiI was almost 100% in vitro, and the SPIO particles with high electron density were mainly distributed in lysosome, mitochondria and other membranous structures in mitochondria, but not in nucleus under transmission electron microscope. The ultrastructure of BMSCs was clear and the morphology was well preserved. Trypan blue exclusion assay showed that there was no significant difference in cell viability between the combined labeled BMSCs group and the unlabeled BMSCs group. It was found that there was no significant difference in cell proliferation between the combined labeled BMSCs group and the unlabeled BMSCs group. Combined labeling of BMSCs by dexamethasone, After induction of insulin 3-isobutyl -1-methylxanthine and indomethacin in vitro, lipid droplets in the cytoplasm were observed under transmission electron microscope, and BMSCs labeled by 5-azacytidine was induced in vitro. The positive expression of cardiac troponin TcTnT was identified by immunofluorescence. Conclusion the combined labeling rate of bone marrow mesenchymal stem cells (BMSCs) in rabbits was higher than that of CM-DiI, and had no effect on survival, proliferation and differentiation.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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