CM-DiI标记大鼠肾脂肪囊来源脂肪间充质干细胞的可行性研究

发布时间：2018-06-02 17:00

本文选题：肾脂肪囊 + 脂肪间充质干细胞　；参考：《辽宁医学院》2012年硕士论文

【摘要】：目的探讨荧光染料氯甲基苯甲酰氨(chlormethyl-benzamidodialkylcarbocyanine,CM-DiI)对大鼠肾脂肪囊来源脂肪间充质干细胞(Kidney fat capsule adipose-derived mesenchymal stem cells,KFC-ADSCs)的标记效果，并分析评价该标记方法对KFC-ADSCs的细胞形态、生长及增殖能力等生物学特性的影响，旨在为KFC-ADSCs移植的体内示踪与归巢研究寻找一种实用而有效的细胞标记方法。方法采用胶原酶消化法分离、培养SD大鼠KFC-ADSCs；流式细胞仪检测KFC-ADSCs表面标记物；无菌、避光条件下采用CM-DiI细胞标记液标记大鼠第3代KFC-ADSCs，倒置荧光显微镜下观察、计算细胞的标记率及标记的持续时间；MTT法分析比较CM-DiI标记液对KFC-ADSCs的细胞形态、生长及增殖等生物学特性是否存在影响。结果流式细胞仪检测KFC-ADSCs细胞表面标记：间充质干细胞表面相关抗原CD29、CD44呈强阳性表达，内皮细胞表面相关抗原CD31呈阴性表达。倒置荧光显微镜下观察CM-DiI标记的KFC-ADSCs细胞膜及细胞浆被染成红色，细胞核不着色。观察CM-DiI标记的KFC-ADSCs细胞标记率达95%以上，，比较KFC-ADSCs标记前后细胞的形态、生长及增殖等生物学特性无明显改变。CM-DiI标记的KFC-ADSCs经多次传代培养后，红色荧光细胞数量及强度有所下降，但细胞传代至第8代、细胞标记的第15d，CM-DiI细胞标记率仍可达50%以上。结论 CM-DiI可以有效、稳定的标记KFC-ADSCs，标记方法简单，标记效率高，对KFC-ADSCs无毒性作用，适用于KFC-ADSCs的中、短期标记及KFC-ADSCs移植的体内示踪观察。
[Abstract]:Purpose To investigate the labeling effect of fluorescent dye chlormethyl-benzamidalkylcarbocyanine (CM-DiI) on adipose mesenchymal stem cells (KFC-ADSCs) derived from renal fat sac in rats, and to analyze and evaluate the cell morphology of KFC-ADSCs by this method. The purpose of this study is to find a practical and effective method for the study of KFC-ADSCs transplantation in vivo for tracing and homing. Method KFC-ADSCs of SD rats were isolated by collagenase digestion, KFC-ADSCs surface markers were detected by flow cytometry, KFC-ADSCswere labeled with CM-DiI cell labeled solution in the condition of sterility, and observed under inverted fluorescence microscope. The cell labeling rate and the labeling duration were calculated. The effects of CM-DiI labeling solution on the cell morphology, growth and proliferation of KFC-ADSCs were compared. Result The surface markers of KFC-ADSCs cells were detected by flow cytometry: CD29 CD44 was strongly expressed on mesenchymal stem cells and CD31 was negative on endothelial cells. The CM-DiI labeled KFC-ADSCs cell membrane and cytoplasm were stained red and the nucleus was not stained under inverted fluorescence microscope. The labeling rate of KFC-ADSCs cells labeled with CM-DiI was more than 95%. The number and intensity of red fluorescent cells decreased after repeated passage culture of KFC-ADSCs labeled with KFC-ADSCs. However, the labeling rate of CM-DiI cells was still over 50% on the 15th day after passage to the 8th passage. Conclusion CM-DiI can effectively and stably label KFC-ADSCs.The labeling method is simple, the labeling efficiency is high, and it has no toxic effect on KFC-ADSCs. It is suitable for the in vivo tracer observation of middle and short term labeling of KFC-ADSCs and KFC-ADSCs transplantation.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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