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目的该研究拟探讨体外培养的永生型兔骨髓间质干细胞(rBMMSCs)是否有分化为脂肪细胞的能力以及分化过程中过氧化物增殖激活受体(peroxisome proliferator activiated receptor,PPAR)和脂蛋白脂酶(lipoprotein lipase,LPL)表达的变化。方法无菌条件下提取兔股骨骨髓,采用密度梯度离心法分离兔骨髓间质干细胞;采用油红O染色检测细胞内脂滴;采用RT-PCR检测PPARγ和LPL mRNA的表达。结果反复传代4个月后永生型rBMMSCs仍然具有稳定的增殖能力,成脂诱导21d后油红O染色显示有(70.2±8.2)%的永生型rBMMSCs分化成脂肪细胞,RT-PCR检测表明PPARγ和LPL mRNA分别在第6天和第7天表达突然增强,成脂诱导第14天PPARγ表达比第7天显著增强。结论采用本方法提取的rBMMSCs在体外培养4个月后仍具有稳定的增殖和分化为脂肪细胞的能力。本方法简易可行,本法所提取的细胞可作为研究脂肪细胞早期分化的理想的实验模型。
Objective To investigate whether the immortalized rabbit bone marrow mesenchymal stem cells (rBMMSCs) cultured in vitro have the ability to differentiate into adipocytes and the peroxisome proliferator activated receptor (PPAR) and lipoprotein lipase (lipoprotein lipase, LPL) expression changes. Methods Rabbit femur bone marrow was extracted under aseptic conditions. Rabbit bone marrow mesenchymal stem cells were isolated by density gradient centrifugation. Lipid droplet was detected by oil red O staining. The expression of PPARγ and LPL mRNA was detected by RT-PCR. Results Immortalized rBMMSCs still had stable proliferative capacity after repeated passages for 4 months. Oil-red O staining showed (70.2 ± 8.2)% of immortalized rBMMSCs differentiated into adipocytes 21 days after adipogenic induction. RT-PCR showed that PPARγ and LPL mRNA expression increased suddenly on the 6th and the 7th day, respectively, while PPARγ expression increased significantly on the 14th day after adipogenic induction than on the 7th day. Conclusion The rBMMSCs extracted by this method still have the ability of stable proliferation and differentiation into adipocytes after cultured in vitro for 4 months. The method is simple and feasible, and the cells extracted by the method can be used as an ideal experimental model for studying the early differentiation of adipocytes.