兔脂肪干细胞体外培养后改良诱导成软骨细胞的实验研究
发布时间：2018-10-19 16:31
【摘要】：目的:探索兔脂肪组织中分离、培养及鉴定干细胞的方法;观察体外长期培养对新西兰大白兔脂肪间充质干细胞(Adipose-Derived Stem Cells,ADSCs)的基本生物学性状及定向改良诱导分化为软骨细胞的能力。方法:在无菌条件下从雄性大白兔颈部区取皮下脂肪组织约5～10g,I型胶原酶消化后,1500r/min离心10min,待细胞长满瓶底80%时用2.5g/L胰蛋白酶消化,按1:3比例传代培养、鉴定,至第3代时加入软骨诱导培养基14d后通过免疫荧光和RT-PCR对其相关表型进行检测,阿新蓝染色及II型胶原免疫组织化学检测脂肪间充质干细胞向软骨细胞的诱导分化。结果:兔脂肪间充质干细胞形态:原代及传代的兔脂肪干细胞均呈长梭形或多边形贴壁生长,生长分化活跃。兔脂肪间充质干细胞的生长曲线:传代的兔脂肪间充质干细胞生长曲线呈“S”形。新西兰大白兔脂肪组织分离培养出的细胞经流式细胞仪检测CD44、CD49d表达阳性,CD45表达阴性。加入软骨诱导培养基定向诱导72h后,细胞生长缓慢,由长梭形逐渐变成圆形,并渐渐有软骨结节形成,胞浆中出现棕色颗粒,细胞表面有较多伪足,具备正常细胞器结构。诱导分化2周后,荧光免疫检测有特异软骨基质Ⅱ型胶原和蛋白聚糖的表达, RT- PCR检测显示有Ⅱ型胶原、Aggrecan和Sox- 9 mRNA的表达,阿新蓝染色及II型胶原免疫组织化学检测阳性。结论:新西兰大白兔脂肪组织能够分离培养出脂肪干细胞,兔源性脂肪干细胞体外以成纤维细胞样形态贴壁生长,在体外具有生长稳定,增殖较快的特点,生物学特性与骨髓间充质干细胞相似,能向软骨细胞分化,在体外长期培养后,其一般生物学性状可保持稳定。
[Abstract]:Objective: to explore the method of isolation, culture and identification of stem cells in rabbit adipose tissue. To observe the basic biological characteristics of adipose mesenchymal stem cells (Adipose-Derived Stem Cells,ADSCs) of New Zealand white rabbits and their ability to differentiate into chondrocytes in vitro. Methods: the subcutaneous adipose tissue of male white rabbits was digested with subcutaneous adipose tissue of about 5U 10g / L collagenase under aseptic condition and centrifuged by 1500r/min for 10 mins. The cells were digested with 2.5g/L trypsin when the cells grew to the bottom of the bottle and cultured at 1:3. The phenotypes were detected by immunofluorescence and RT-PCR, and the differentiation of adipose mesenchymal stem cells into chondrocytes was detected by Arxin blue staining and II collagen immunohistochemistry. Results: the morphology of rabbit adipose mesenchymal stem cells: primary and passage of rabbit adipose stem cells were long fusiform or polygonal adherent growth and active growth and differentiation. The growth curve of rabbit adipose mesenchymal stem cells: the growth curve of rabbit adipose mesenchymal stem cells was "S" shape. The positive expression of CD44,CD49d and negative expression of CD45 were detected by flow cytometry in adipose tissue isolated from New Zealand white rabbit. After 72 hours of induction in cartilage induction medium, the cells grew slowly, gradually became round from long spindle shape, and gradually formed cartilage nodules, brown granules appeared in the cytoplasm, and the cell surface had more pseudopodia, with normal organelle structure. After 2 weeks of differentiation, the expression of type 鈪，
本文编号：2281697
[Abstract]:Objective: to explore the method of isolation, culture and identification of stem cells in rabbit adipose tissue. To observe the basic biological characteristics of adipose mesenchymal stem cells (Adipose-Derived Stem Cells,ADSCs) of New Zealand white rabbits and their ability to differentiate into chondrocytes in vitro. Methods: the subcutaneous adipose tissue of male white rabbits was digested with subcutaneous adipose tissue of about 5U 10g / L collagenase under aseptic condition and centrifuged by 1500r/min for 10 mins. The cells were digested with 2.5g/L trypsin when the cells grew to the bottom of the bottle and cultured at 1:3. The phenotypes were detected by immunofluorescence and RT-PCR, and the differentiation of adipose mesenchymal stem cells into chondrocytes was detected by Arxin blue staining and II collagen immunohistochemistry. Results: the morphology of rabbit adipose mesenchymal stem cells: primary and passage of rabbit adipose stem cells were long fusiform or polygonal adherent growth and active growth and differentiation. The growth curve of rabbit adipose mesenchymal stem cells: the growth curve of rabbit adipose mesenchymal stem cells was "S" shape. The positive expression of CD44,CD49d and negative expression of CD45 were detected by flow cytometry in adipose tissue isolated from New Zealand white rabbit. After 72 hours of induction in cartilage induction medium, the cells grew slowly, gradually became round from long spindle shape, and gradually formed cartilage nodules, brown granules appeared in the cytoplasm, and the cell surface had more pseudopodia, with normal organelle structure. After 2 weeks of differentiation, the expression of type 鈪，
本文编号：2281697
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