犊牛脂肪干细胞的分离、培养与鉴定

为给奶牛营养代谢病等相关研究提供种子细胞,本试验从犊牛肾周脂肪组织中分离出脂肪干细胞(adipose-derived stem cells, ADSCs),采用组织剪碎法结合0.25%Ⅰ型胶原酶消化法消化、收集原代犊牛ADSCs,通过细胞形态学观察,使用免疫荧光染色法检测第5代ADSCs表面特异性蛋白,并使用CCK-8法测定第5、10代细胞生长曲线,观察第10代细胞的成脂、成骨分化能力。结果显示,分离出的犊牛ADSCs接种4 d后细胞呈成纤维细胞样生长;免疫荧光染色结果显示,分离的细胞Vimentin、CD34、CD44、CD90呈阳性,CD31、CD45呈阴性;该细胞在适当的诱导条件下可以定向分化为脂肪细胞和成骨细胞。本试验分离培养的细胞是犊牛脂肪干细胞,具有成脂、成骨分化能力。

Isolation,culture and identification of adipose-derived stem cells from calves

In order to provide seed cells for related research on vegetative metabolic diseases of cows, adipose-derived stem cells (ADSCs) were eparated from perirenal adipose tissue of calves. ADSCs were collected by organization minced and 0.25% collagenase Ⅰ digestion by different times. The separated cells were proved by the following aspects, observed the morphology, passage 5 of ADSCs were used to detect the surface-specific proteins, passage 5 and 10 were used to detect the growth curve by CCK-8, passage 10 were used to detect the ability of adipogenic and osteogenic differentiation. The results showed that the ADSCs from calves were grown as fibroblast-like after 4 days of inoculation. Immunofluorescence staining showed that the isolated cells expressed Vimentin, CD34, CD44, and CD90, but did not express CD31 and CD45. The cells can be differentiated into adipocytes and osteoblasts under appropriate induction conditions. The separated cells were ADSCs with the ability of adipogenic and osteogenic differentiation.