快速诱导大鼠骨髓间充质干细胞成脂分化的培养体系

In vitro rapid culture system to induce the adipogenic differentiation of rat bone marrow mesenchymal stem cells

背景:目前常用的成脂刺激剂主要由胰岛素、地塞米松、IBMX和吲哚美辛组成,该体系涉及处理因素多、诱导周期长且对细胞毒性大,不利于脂肪形成抑制效应的研究。目的:建立快速诱导大鼠骨髓间充质干细胞成脂分化的培养体系。方法:大鼠全骨髓细胞原代培养,胰酶消化传代,富集形态均质的间充质干细胞,利用过氧化物酶体增殖物活化受体γ激动剂噻唑烷二酮类药物罗格列酮和吡咯列酮体外单独诱导大鼠骨髓间充质干细胞成脂分化,设立无糖、低糖和高糖DMEM三种基础培养体系,以经典的成脂刺激剂作为阳性对照,在诱导的不同时间点对分化细胞进行形态学观察和油红O染色。结果与结论:与经典成脂刺激剂相比,罗格列酮或吡咯列酮单独均能诱导大鼠骨髓间充质干细胞向脂肪细胞分化,吡咯列酮诱导48h和罗格列酮诱导72h均可观察到富含脂滴或脂泡以及油红O染色的阳性细胞,吡咯列酮与罗格列酮的最佳诱导浓度分别为0.125mmol/L和10μmol/L,而高糖环境利于大鼠骨髓间充质干细胞成脂分化。说明基于高糖培养环境的以吡咯列酮或罗格列酮做为成脂刺激剂的培养体系可快速诱导大鼠骨髓间充质干细胞向脂肪细胞分化。

BACKGROUND：The commonly used adipogenic irritants consist of insulin, dexamethasone, 3-isobutyl-1-methylxanthine and indomethacin. The system is not conducive to the study of adipogenesis inhibitory effect due to the disadvantages of multi-processing factors, long induction period and great cytotoxicity. OBJECTIVE：To develop a rapid culture method to induce adipogenic differentiation of rat bone marrow mesenchymal stem cel s. METHODS：Rat whole bone marrow cel s were primary cultured and passaged with trypsin digestion method. The homogeneous mesenchymal stem cel s were enriched, and adipogenic differentiation of rat bone marrow mesenchymal stem cel s were in vitro induced with thiazolidinediones （rosiglitazone and pioglitazone）, the agonists of peroxisome proliferator-activated receptorγ. Glucose-free, low glucose and high glucose Dulbecco＇s modified Eagle＇s media were established, and the classic adipogenic irritants were as the positive control. Morphological observation and oil red O staining were performed to observe the differentiated cel s at different time points. RESULTS AND CONCLUSION：Compared with the classic adipogenic irritants, the rosiglitazone and pioglitazone could induce the rat bone marrow mesenchymal stem cel s to differentiate into adipocytes, and accumulations of lipid droplets or lipid vesicles and oil red O staining positive cel s could be observed after induced with pioglitazone for 48 hours and rosiglitazone for 72 hours. The optional concentration of pioglitazone and rosiglitazone for adipogenic induction was 0.125 mmol/L and 10μmol/L, respectively. High glucose could enhance the adipogenic differentiation of rat bone marrow mesenchymal stem cel s. The culture system with the adipogenic irritants of pioglitazone and rosiglitazone under the high glucose environment can rapidly induce the rat bone marrow mesenchymal stem cel s to differentiate into adipocytes.