15d-PGJ2对骨髓间充质干细胞培养上清中细胞因子的影响  

Effect of 15-deoxy-Δ^(12,14)-prostaglandin J_2 on the Cytokines in the Culture Supernatant of Bone Marrow Mesenchymal Stem Cells

作　　者：池颖 杜文静 崔俊杰 陈芳 韩之波 马凤霞 李雪 杨少光 卢士红 韩忠朝 

机构地区：[1]中国医学科学院、北京协和医学院血液学研究所、血液病医院实验血液学国家重点实验室,天津300020

出　　处：《中国实验血液学杂志》2013年第6期1557-1562,共6页Journal of Experimental Hematology

基　　金：国家重点基础研究发展计划"973计划"(编号2011CB964800);国家高技术研究发展计划"863计划"(编号2011AA020118)

摘　　要：PPARγ配体15d-PGJ2(15-deoxy-Δ12,14-prostaglandin J2)对细胞有抑制增殖、促进分化和凋亡的作用,但是它对骨髓间充质干细胞(BM-MSC)培养上清中细胞因子的影响还未见报道。本研究探讨15d-PGJ2对BM-MSC培养上清中细胞因子的影响。首先对正常人骨髓进行分离培养获得可传代的贴壁生长的成纤维细胞样细胞,然后应用流式细胞术检测细胞的表型,应用条件培养液诱导细胞向成脂和成骨分化以鉴定所获得细胞为BM-MSC。在BM-MSC培养体系中加入10、20、40和60μmol/L的15d-PGJ2并培养24 h,用实时定量PCR测定PPARγmRNA水平,共聚焦免疫荧光技术检测PPARγ的表达水平。结果发现,当15d-PGJ2的浓度达到20μmol/L时细胞出现了凋亡并大量从培养瓶表面脱落;而10μmol/L的15d-PGJ2刺激细胞24 h后PPARγ的mRNA表达水平增高,与对照组相比有显著性差异(P<0.05)。然后用含有10μmol/L 15d-PGJ2和不含15d-PGJ2体系分别培养BM-MSC 24 h并用蛋白芯片检测培养上清,发现有部分因子表达水平发生了变化。结论:TIMP-2在15d-PGJ2刺激后下调,且信号值及变化水平有意义。15-Deoxy-△12,14-prostaglandin J2 （15d-PGJ2）, a well known peroxisome proliferator activated receptor （PPAR） γligand, has been shown to inhibit cellular proliferation and induce apoptosis and differentiation. However, whether 15d-PGJ2 influences the cytokines in the culture supernatant of bone marrow mesenchymal stem cells （ BM- MSC） is unknown. This study was purposed to investigate the influence of 15d-PGJ2 on cytokines in the culture superna- tant of BM-MSC. The flbroblast-like cells attached to the culture dish from bone marrow of healthy donors were isola- ted. The immunophenotype and differentiation potential of the obtained cells were detected by flow cytonetrcy and oil red O and von kassa staining respectively to connfirm that these cells were BM-MSC. Thereafter, the BM-MSC were cultured with complete medium supplemented with 10, 20, 40 and 60 μmol/L 15d-PGJ： for 24 hours respectively. The real-time PCR was used to assay the PPARγ mRNA level, the confocal immuno fluorescence technique was used to detect the ex- pression level of PPARγ. The results showed that the BM-MSC underwent apoptosis and got detached from the culture dish when the concentration of 15d-PGJ2 was no less than 20μmol/L. The PPARγ mRNA level of BM-MSCs cultured with medium containing 10 μmol/L 15d-PGJ2 was higher than that cultured without 15d-PGJ2, and the difference was statistically significant （P 〈 0.05）. The enhancement of PPARγ expression was observed after stimulated by 15d-PGJ2. The protein chip detecting the culture supematants of BM-MSC cultured with 10 μmol/L 15d-PGJ2 or without 15d-PGJ2 for 24 hours demonstraded that expression levels of some of the cytokines varied. It is concluded that the down-regulation of TIMP-2 exists after treatment of 15d-PGJ2, which is statistical significant.

关 键 词：15D-PGJ2 间充质干细胞 细胞因子 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]