骨髓源性脂肪祖细胞培养及生物学特性研究  

Study on culture and biological characteristics of BMAPCs

作　　者：朱光旭[1] 王金祥[1] 周芳[1,2] 李自安[1] 刘菊芬[1] 阮光萍[1] 白盈盈[1,2] 潘兴华[1] 

机构地区：[1]成都军区昆明总医院细胞生物治疗中心、干细胞与免疫细胞生物医药技术国家地方联合实验室、云南省细胞治疗技术转化医学重点实验室,昆明650032 [2]昆明医科大学成都军区昆明总医院临床学院

出　　处：《西南国防医药》2016年第12期1386-1391,共6页Medical Journal of National Defending Forces in Southwest China

基　　金：国家科技支撑计划项目(2014BI01B00);国家自然科学基金(81170316);云南省科技计划项目(20111HB050,2013DA004)

摘　　要：目的体外培养骨髓脂肪祖细胞（BMAPs），比较其与骨髓间充质干细胞（BMSCs）在形态、表型特征、成脂肪分化能力以及脂肪分化相关基因表达方面的差异。方法无菌取1-2月龄雄性SD大鼠的股骨和胫骨骨髓制作细胞悬液，EBM-2（含20％FBS、15μg／ml ECGs）培养基差速贴壁法培养第3次贴壁细胞，取传代至第3-5代细胞用于实验。流式细胞分析检测CD34、CD44、CD45及CD90表达；STEMPRO Adipogenesis Differentiation Kit检测细胞成脂肪分化能力；实时荧光定量聚合酶链式反应（real-timeq PCR）检测过氧化物酶体增殖激活受体γ（Ppaγ）、脂肪酸结合蛋白4（Fabp4）、脂蛋白脂肪酶（Lpl）、CCAAT／增强子结合蛋白α（cebpa）、葡萄糖转运蛋白4（Slc2a4）及前脂肪细胞因子1（preadl）mRNA表达水平。结果传代接种培养24h后，BMSCs形态上呈宽大、扁平状，而BMAPs大部呈长三角状；接种48h后，BMSCs排列更为有序，而BMAPs多交织成网状。BMAP表达CD34、CD44、CD45及CD90，BMSC表达CD44及CD90，未检测到CD34和CD45表达。诱导培养后第4d，BMAPs成脂率显著高于BMSCs（P〈0．01）；到第8d。BMAPs成脂率为（87．2±1L67）％，BMSCs成脂率为（18．5±10．2）％，差异具有统计学意义（P〈0．01）。诱导分化后第4、8d，Cebpa、Fabp4、Lp1、Ppaγ和Slc2a4mRNA在BMAPs表达水平均显著高于BMSCs（P〈0．01）。结论本实验获得了1种具有干细胞的生物学特性，高表达脂肪分化相关的特异性基因，可高效均质性地分化为脂肪细胞的BMAPs。Objective To explore the difference in morphology, phenotypic characteristics, capacity to differentiate into fat cells and expressions of genes relevant to differentiation into fat cells between bone marrow adipocyte progenitor cells （BMAPCs） and bone mesenchymal stem ceils （BMSCs）. Methods The femoral and tibial bone marrow of male SD rats aged one to two months was sampled in a sterile manner to prepare cell suspension. The third adherent cells were cultivated by differential adhesion method in EBM-2 （containing 20% FBS, 15 μg/ml ECGs） culture media. Passage three（P3）-passage five （PS） of the third adherent cells were used in the experiments. The expressions of CD34, CD44, CD45 and CD90 were detected by flow cytometry; the capacity of cells to differentiate into fat cells was detected by STEMPRO Adipogenesis Differentiation Kit. The levels of Ppaγ, Fabp4, Lp1, CCAAT/enhancer binding protein α（Cebpa）, glucose transporter （GLUT） 4 （Slc2a4） and preadl mRNA were detected by real-time qPCR. Results 24 hours after the passage inoculation culture, BMSCs were wide and flat in morphology, while the most of BMAPCs were in long triangle shape. 48 hours after the inoculation, the arrangement of BMSCs was more orderly, while most BMAPCs were interwoven into a network. BMAPCs expressed CD34, CD44, CD45 and CD90; BMSCs expressed CD44 and CD90, CD34 and CD45 were not expressed. On the fourth day after the induction culture, the fat cell percentage of BMAPCs was significantly higher than that of BMSCs （P 〈 0.01）; on the eighth day, the fat cell percentage of BMAPCs was（87.2±11.67）%, while that of BMSCs was （18.5±10.2）%, showing a more significantly difference（P 〈 0.01）. On the fourth and eighth day after the induction differentiation, the levels of Cebpa, Fabp4, Lp1, Ppa3＇ and Slc2a4 mRNA in BMAPCs were significantly higher than those in BMSCs （P〈 0.01）. Conclusion One kind of BMAPCs which have the biological characteristics of stem ceils and high levels of dist

关 键 词：骨髓 脂肪祖细胞 间充质干细胞 脂肪细胞 分化 

分 类 号：R318.1[医药卫生—生物医学工程]