作　　者： (季伟）; (李海峰）; (程实）; (陈佳海）; (徐成）; (刘玥）; (何勍）; (阮狄克）;

机构地区： 第四军医大学西京医院骨科,西安710032 海军总医院骨科,北京100048

出　　处： 《中国骨与关节外科》 2017年第2期148-153,108,共7页

摘　　要： 背景:对于骨髓间充质干细胞(BMSCs)与髓核(NP)细胞共培养的研究主要集中在不同条件下的干细胞向NP细胞表型的分化。一些研究表明,在动物试验中干细胞影响退变的NP细胞。少有研究人BMSCs对NP细胞的影响,特别是在低氧条件下。目的:建立同一患者的BMSCs和NP细胞的共培养模型,探讨在低氧环境下BMSCs对NP细胞的生物学影响。方法:分离培养患者的NP细胞和BMSCs。并将其分为两组。A组为NP细胞单独培养组,B组为NP细胞与BMSCs共培养组。两组细胞均连续培养14 d。通过光学显微镜观察两组细胞形态及数量,用流式细胞仪和扫描电子显微镜(SEM)观察细胞死亡。用细胞计数kit-8试剂盒(CCK8)评估细胞增殖,进一步采用实时/逆转录-聚合酶链反应(RT-PCR)检测细胞外基质(ECM)和NP细胞特殊表型的标记的表达。结果:培养14 d后,光学显微镜下观察,单独培养组可见NP细胞贴壁和延伸,NP细胞的数量比培养前多。在共培养组,NP细胞形态是纺锤形,贴壁、延伸比单培养组好,NP细胞的数量远远超过培养前,并且多于单独培养组。CCK8检测表明,培养14 d后,与单独培养组相比,共培养组的NP细胞更多(P<0.05),单独培养组细胞凋亡率高于共培养组(P<0.05)。在低氧和与BMSCs共同培养条件下,蛋白多糖(AGG)、胶原Ⅱ(COL2),SOX-9,角蛋白19(KRT19),碳酸酐酶Ⅻ(CA12)和低氧诱导因子(HIF)表达增加。RT-PCR检测表明这些基因和蛋白的合成、分泌在与BMSCs共培养的NP细胞组中明显高于NP细胞单独培养组。结论:在体外更接近体内条件的低氧直接共同培养条件下,BMSCs可能会通过调节ECM和调节基因来保护或激活退化的NP细胞。为BMSCs治疗椎间盘退变提供了一个重要的新思路。 Background：Numerous studies about the co-culture of bone marrow mesenchymal stem cells （BMSCs） with nucleus pulposus （NP） cells have focused on the differentiation of stem cells towards NP cell phenotypes in various conditions. There are few studies on the effects of human BMSCs on the NP cells which are homologous, especially under hypoxia environment. Objective：To establish a co-cultured model of human NP cells with human BMSCs which come from the same patient, so as to explore the biological effect of BMSCs on NP cells under hypoxic environment. Methods：Human NP cells and BMSCs from one patient were isolated and cultured. Then they were divided into two groups. There were only NP cells in group A, and NP cells were co-cultured with BMSCs in group B. All the cells were cultured for 14 d. The NP cells morphology of both groups was observed under optical microscope. Cell apoptosis was evaluated by flow cytometry and scanning electron microscope （SEM）. Cell proliferation was assessed by cell counting kit 8 （CCK8）. Real-time PCR （RT-PCR）, western blot and immunofluorescent staining were used to determine the expression of the extracellular matrix （ECM） and NP cells special phenotype markers. Results：After cultures 14 d, NP cell adherence and elongation were found in group A under optical microscope, and the amount of the NP cells increased. The NP cells were spindle-shaped in group B. The NP cell adherence and elongation in group B were superior to that in group A, and the amount of the NP cells in group B were significantly greater than that in group A. The rate of cell apoptosis in group A was significantly higher than that in group B （P〈0.05）. The expression of proteoglycan （AGG）, collagenⅡ（COL2）, SOX-9, keratin 19 （KRT19） and carbonic anhydraseⅫ（CA12） and hypoxia-inducible factor （HIF） increased under co-culture and hypoxic environment. RT-PCR showed that the synthesis and secretion of these genes and proteins in group B were significantly higher tha

关 键 词： 共培养 低氧 骨髓间充质干细胞 髓核细胞 椎间盘 细胞治疗