大鼠脂肪干细胞的分离培养鉴定及多向诱导分化研究

发布时间：2018-04-20 10:29

本文选题：脂肪干细胞 + 大鼠　；参考：《中华中医药学刊》2017年10期

【摘要】：目的:通过体外分离培养脂肪干细胞,并进行传代、表型鉴定及多向诱导分化,进一步了解其生物学特性,为临床运用奠定实验基础。方法:取SD大鼠附睾近心端,肾周及腹股沟韧带皮下脂肪组织,采用密度梯度离心和贴壁培养相结合方法,对脂肪干细胞进行分离、体外培养及扩增、表型鉴定及成骨成脂诱导分化,在倒置显微镜下观察原代及传代细胞形态变化,并采用MTT法检测第3、5、7、9代细胞增殖能力,用流式细胞仪检测细胞表面抗原标记,取第3代细胞进行成骨成脂诱导分化,并采用茜素红染色、油红O染色方法进行鉴定。结果:采用密度梯度离心和贴壁培养相结合方法得到的脂肪干细胞,其细胞形态较为均一,基本呈长梭形,旋涡状生长,并形成集落样生长。MTT法检测第3、5、7、9代ADSCs呈典型的"S"形增殖曲线,符合干细胞的特性,脂肪干细胞表型鉴定结果显示CD29阳性高表达,CD34阴性表达,CD45低表达,CD105弱阳性表达,脂肪干细胞能向成骨细胞及脂肪细胞分化。结论:脂肪干细胞取材较方便,含量丰富、并能够在体外分离培养,其稳定表达特异性的表面抗原,并且经过相应诱导培养后可向成骨及成脂多向分化,可作为优良的骨组织工程的种子细胞来源。
[Abstract]:Objective: to investigate the biological characteristics of adipose stem cells (ASCs) and to establish the experimental basis for clinical application by means of isolation and culture in vitro, passage, phenotypic identification and multidirectional differentiation. Methods: the adipose tissues of the proximal epididymis, perirenal and inguinal ligaments of SD rats were isolated, cultured and expanded in vitro by density gradient centrifugation and adherent culture. Phenotypic identification and osteogenic adipogenic differentiation were used to observe the morphological changes of primary and passage cells under inverted microscope. MTT assay was used to detect the proliferative ability of passage 3 ~ (5) and 7 ~ (th) passage cells, and flow cytometry was used to detect cell surface antigen labeling. Osteogenic adipogenic differentiation of the third passage cells was carried out and identified by alizarin red staining and oil red O staining. Results: the adipose stem cells were obtained by density gradient centrifugation and adherent culture. The results showed that the proliferation curve of ADSCs in the 3rd generation was "S" shape, which was in line with the characteristics of stem cells. The phenotypic identification of adipose stem cells showed that the positive expression of CD29 was high, the negative expression of CD34 was negative, the expression of CD45 was low and the expression of CD105 was weak. Adipose stem cells can differentiate into osteoblasts and adipocytes. Conclusion: adipose stem cells are convenient and rich in content, and can be isolated and cultured in vitro. They can express specific surface antigen stably, and can differentiate into osteogenesis and adipogenesis after induction and culture. It can be used as a good seed cell source for bone tissue engineering.
【作者单位】：浙江中医药大学附属第一医院骨伤科;浙江中医药大学附属第三医院骨伤科;
【基金】：国家自然科学基金项目(81273771) 浙江省姚新苗名老中医专家传承工作室建设项目浙江省中医药重点学科(中医老年骨伤学)建设项目(2012-XK-A17) 浙江省中医药管理局一般项目(2016ZA103) 浙江省科技厅创新团队项目(2011R50022-09) 浙江中医药大学项目(2015XJ05-01) 浙江中医药大学附属第三医院项目(2015ZS03-01)
【分类号】：R580;R683

【相似文献】