神经营养因子3增强脂多糖刺激的人骨髓间充质干细胞的成骨能力  

Neurotrophin-3 enhances the osteogenesis ability of human bone marrow mesenchymal stem cells stimulated by lipopolysaccharide

作　　者：张善强[1] 金海峰[1] 姚立杰[1] 邓凤春[1] 沈雷[1] ZHANG Shanqiang, JIN Haifeng, YAO Lijie, DENG Fengchun, SHEN Lei(Department of Anatomy, Qiqihar Medical College, Qiqihar 161006, Chin)

机构地区：[1]齐齐哈尔医学院基础医学院解剖学教研室,黑龙江齐齐哈尔161006

出　　处：《细胞与分子免疫学杂志》2018年第1期47-52,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：齐齐哈尔市科技局社会发展攻关项目(SFGG-201633);齐齐哈尔医学院博士基金(QY2015B-04)

摘　　要：目的探讨神经营养因子3(NT-3)在炎症环境下对人骨髓间充质干细胞(hBMSC)的保护及促进hBMSC向成骨细胞分化的能力。方法采用脂多糖(LPS)建立细胞炎症模型,未进行任何刺激的hBMSC为炎症对照组;以100 ng/m L人NT-3重组蛋白刺激的hBMSC为NT-3组;若先用100 mmol/L恩波酸吡维铵(PP)作用12 h,再添加100 ng/m L人NT-3重组蛋白刺激者为Wnt抑制剂组,常规条件下培养的hBMSC为正常对照组,每组均进行成骨细胞诱导实验。利用CCK-8法检测hBMSC的增殖情况、异硫氰酸荧光素标记的膜联素Ⅴ/碘化丙啶(annexinⅤ-FITC/PI)双标记结合流式细胞术检测hBMSC的凋亡,ELISA检测hBMSC核心结合因子runt相关转录因子2(RUNX2)、碱性磷酸酶(ALP)含量、茜素红染色检测钙结节形成情况。结果与炎症对照组相比,NT-3组的hBMSC增殖活性明显增强,细胞凋亡显著降低,且NT-3组的RUNX2、ALP等蛋白含量或茜素红染色强度均明显增加。与NT-3组相比,Wnt抑制剂组的hBMSC增殖活性降低,细胞凋亡增加,RUNX2、ALP等蛋白含量或茜素红染色强度均明显降低。结论 NT-3具有保护hBMSC抗炎症损伤的作用,并促进hBMSC向成骨细胞分化。Objective To investigate the protective effect of neurotrophin-3 （NT-3） on human bone marrow mesenchymal stem cells （hBMSCs） and its ability to promote the differentiation of hBMSCs into osteoblasts in the inflammatory environment. Methods The cell inflammation model was established by lipopolysaccharide （LPS）. The hBMSCs without any stimulation was defined as the inflammatory control group; the hBMSCs stimulated by 100 ng/mL human NT-3 recombinant protein as the NT-3 group; the hBMSCs stimulated by 100 mmol/L pyrvinium pamoate （PP） for 12 hours and then stimulated by 100 ng/mL human NT-3 recombinant protein as the Wnt inhibitor group; the normal cultured hBMSCs as the normal control group. We performed the experiment of osteoblast induction on all groups. CCK-8 assay was used to detect the proliferation of hBMSCs; the fluorescein isothiocyanate labeled annexin V/propidium iodide （Annexin V-FITC/PI） double labeling combined with flow cytometry was used to detect the apoptosis of hBMSCs; ELISA was used to detect the protein levels of runt-related transcription factor 2 （RUNX2） protein and alkaline phosphatase （ALP）; and the alizadn red staining experiment was conducted to detect the ability of calcium nodule formation. Results Compared with the inflammatory control group, the proliferative activity of hBMSCs in NT-3 group significantly increased, the apoptosis obviously decreased, and the contents of RUNX2 and ALP, as well as the intensity of alizarin red staining in NT-3 group evidently rose. Compared with the NT-3 group, the proliferative activity of hBMSCs in the Wnt inhibitor group was inhibited, the cell apoptosis was promoted, and the contents of RUNX2 and ALP, as well as the intensity of alizarin red staining in the Wnt inhibitor group were reduced remarkably. Conclusion NT-3 can protect hBMSCs from anti-inflammatory damage and promote the differentiation of hBMSCs into osteoblasts.

关 键 词：神经营养因子3(NT-3) 人骨髓间充质干细胞 成骨分化 炎症 

分 类 号：R392-33[医药卫生—免疫学] Q25[医药卫生—基础医学]