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目的体外定向诱导猴骨髓间充质干细胞(bone marrow mesenhymal stem cells,BMSCs)分化为脂肪细胞。方法抽取2～3岁龄雄性恒河猴髂骨骨髓,应用密度梯度离心法提取单核细胞层进行贴壁培养,获取原代(P0)猴BMSCs后进行传代培养,并进行多向分化能力鉴定。分别取第3代(P3)、第5代(P5)、第8代(P8)猴BMSCs,实验组加入脂肪细胞诱导分化培养液,对照组加入常规培养液。倒置相差显微镜下观察细胞形态变化,诱导3周后行油红O染色,计算脂肪细胞分化率,并比较不同代次之间脂肪细胞分化率之间的差异。结果实验抽取的猴骨髓单核细胞具有成脂及成骨分化能力,从而表明其为多能性干细胞。成脂诱导分化培养后第5天,实验组即可观察到少数细胞胞质内出现脂滴,随后胞质内脂滴数量、大小及含脂滴细胞数量逐渐增加,约3周时达到高峰,随后诱导细胞迅速出现死亡;油红O染色示脂滴呈橙红色;脂肪细胞分化率:P3(48.3±10.0)%,P5(58.7±5.3)%,P8(68.1±5.5)%,差异有统计学意义(P<0.05)。对照组未见脂滴形成,油红O染色呈阴性。结论脂肪细胞诱导分化培养液可以在体外将猴BMSCs定向诱导分化为脂肪细胞,脂肪细胞分化率随着传代次数增加而增加。
Objective To induce bone marrow mesenchymal stem cells (BMSCs) to differentiate into adipocytes in vitro. Methods Bone marrow from 2 to 3-year-old rhesus macaques was collected. The mononuclear cells were isolated from the mononuclear cells by density gradient centrifugation. The primary (P0) monkey BMSCs were obtained and subcultured, and their multi-directional differentiation ability . The third generation (P3), the fifth generation (P5) and the eighth generation (P8) monkey BMSCs were collected. The experimental group was added with adipocyte differentiation culture medium, and the control group was added with routine culture medium. The morphological changes of the cells were observed under an inverted phase contrast microscope. After 3 weeks of induction, the oil red O staining was performed to calculate the adipocyte differentiation rate, and the difference between the adipocyte differentiation rates was compared between different generations. Results The experimental monkey bone marrow mononuclear cells were adipogenic and osteogenic differentiation, indicating that they are pluripotent stem cells. At day 5 after adipogenic differentiation, lipid droplets were observed in the cytoplasm of a few cells in the experimental group, then the number, size and amount of lipid droplets in the cytoplasm increased gradually and reached a peak at about 3 weeks, Then the cells were induced to die quickly. Oil red O staining showed orange-red lipid droplets. The percentage of adipocyte differentiation was P3 (48.3 ± 10.0)%, P5 (58.7 ± 5.3)% and P8 (68.1 ± 5.5)% Significance (P <0.05). No lipid droplet was formed in the control group, and oil red O staining was negative. Conclusion Adipocyte-induced differentiation culture medium can induce the differentiation of monkey BMSCs into adipocytes in vitro. The adipocyte differentiation rate increases with the increase of passage times.