论文部分内容阅读
目的探索TNF-α免疫激活骨髓间充质干细胞(BM-MSC)后,其旁分泌作用对成骨细胞(MG63)功能的影响。方法使用TNF-α预刺激MSCs。制备MSC条件培养基(conditioned medium,MSC-CM)培养成骨细胞(MG63)。CCK8法检测MG63增殖能力变化;细胞划痕法检测MG63迁徙能力变化;Real-time PCR法检测ALP、COLI及OCN基因表达量的变化;免疫荧光法检测OPN表达量变化;茜素红染色检测矿化能力变化。结果 TNF-α预刺激干细胞后,MSC旁分泌功能发生显著变化,其中IL-6、VEGF和TGF-β的分泌量明显升高(P<0.01),但HGF的分泌量无明显变化(P>0.05)。在TNF-α组的MSC-CM作用下,MG63增殖速度加快,细胞划痕结果显示其迁徙能力提高,诱导第4、7天后成骨细胞分化相关基因表达量增多(P<0.05),免疫荧光染色显示TNF-α组的OPN阳性率升高,茜素红染色显示MG63矿化沉积作用增强。结论免疫激活后MSC的旁分泌作用可以显著促进成骨细胞的增殖、迁徙、分化和矿化能力,从多个阶段加强成骨细胞的骨化作用,为临床应用MSC治疗骨质缺损指导了新方向。
Objective To explore the effect of paracrine on the function of osteoblasts (MG63) induced by TNF-α immunostimulating bone marrow mesenchymal stem cells (BM-MSCs). Methods The MSCs were pre-stimulated with TNF-α. Osteoblasts (MG63) were cultured with MSC conditioned medium (MSC-CM). CCK8 assay was used to detect the proliferation of MG63. The cell viability was measured by cell scratch assay. The expression of ALP, COLI and OCN was detected by Real-time PCR. The expression of OPN was detected by immunofluorescence assay. Change in ability. Results The secretion of IL-6, VEGF and TGF-β were significantly increased after TNF-α pre-stimulated stem cells (P <0.01), but there was no significant change in the secretion of HGF (P> 0.05). Under the action of MSC-CM, the proliferation of MG63 was accelerated in TNF-|Á group. The cell scratch showed that the migration ability of MG63 was enhanced. The expression of osteoblast differentiation-related genes increased on the 4th and 7th day after induction (P <0.05) Staining showed that the positive rate of OPN in TNF-α group was increased, alizarin red staining showed that the mineralization of MG63 increased. CONCLUSION: The paracrine effect of MSC after immunostimulation can significantly promote the proliferation, migration, differentiation and mineralization of osteoblasts, enhance the ossification of osteoblasts from multiple stages and guide the new treatment of bone defects by clinical application of MSCs direction.