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目的分离、培养增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)转基因小鼠脂肪间充质干细胞(adipose-derived mesenchymal stem cells,ADMSCs),并研究ADMSC在体外定向诱导下的多向分化潜能。方法分离EGFP转基因小鼠腹股沟的皮下脂肪组织,剪碎后胶原酶消化,含10%优等胎牛血清(fetal calf serum,FBS)的α-MEM培养液培养,取第3代EGFP-ADMSCs进行成骨、成脂诱导,并分别采用茜素红钙盐染色和油红O染色进行鉴定。结果成功获得了稳定表达EGFP的ADMSC;EGFP-ADMSCs经成骨诱导21 d后茜素红染色可见橘红色钙盐沉积,经成脂诱导14 d后油红O染色阳性,并且分化后细胞的EGFP表达不受影响。结论 EGFP转基因小鼠来源的脂肪间充质干细胞具有普通小鼠ADMSCs相同的自我更新增殖和多向分化的干细胞特性,并且能稳定表达EGFP,可以成为研究干细胞修复受损组织的作用机制的有效示踪工具。
OBJECTIVE To isolate and culture adipose-derived mesenchymal stem cells (ADMSCs) of enhanced green fluorescent protein (EGFP) transgenic mice and investigate the multi-directional differentiation potential of ADMSCs induced by in vitro targeting. Methods The subcutaneous adipose tissue of groin in EGFP transgenic mice was isolated and digested with collagenase and then cultured in α-MEM containing 10% fetal calf serum (FBS). The third passage of EGFP-ADMSCs was harvested Bone, adipogenesis, and were identified by alizarin red calcium salt staining and oil red O staining. RESULTS: ADMSCs stably expressing EGFP were successfully obtained. On days 21 after osteogenic induction, EGFP-ADMSCs were stained with alizarin red and showed the deposition of orange-red calcium. After 14 days of adipogenic induction, the oily red O staining was positive. The differentiated EGFP Expression is not affected. CONCLUSIONS: Adipose-derived mesenchymal stem cells derived from EGFP transgenic mice have the same self-renewal and multidifferentiated stem cell characteristics as ADMSCs in mice and their stable expression of EGFP can be an effective indicator for studying the mechanism of stem cell repair in damaged tissues Tracing tool.