动态压应力与共培养微环境对脂肪间充质干细胞增殖及向髓核样细胞分化的影响

发布时间：2018-08-03 07:11

【摘要】：【目的】探讨动态压应力与共培养微环境条件对大鼠脂肪间充质干细胞（ADSCs）增殖及向髓核样细胞分化的影响。 【方法】取原代SD大鼠ADSCs体外培养并扩增，流式细胞仪鉴定细胞表面标志物后，以第三代ADSCs经可控细胞动态压应力加载装置(90mmHg，12h/d)行刺激，并设置阴性组，采用CFSE染色法测细胞增殖活性，分别于染色后24h,48h,72h，96h以倒置荧光显微镜观察并以流式细胞仪检测荧光衰减度。同时将包裹有SD大鼠髓核细胞（NPCs）的藻酸盐微球与第三代ADSCs混合共培养，按细胞共培养和压应力刺激与否分为单纯ADSCs培养组、ADSCs加压组、单纯NPCs培养组、NPCs加压组、共培养组和共培养联合压力组，于干预至第7d时采用RealtimePCR和WesternBlot检测各组贴壁细胞SOX-9，Ⅱ型胶原及aggrecan的表达差异。 【结果】实验所用ADSCs经流式细胞仪鉴定显示CD29、CD44阳性率分别为99.72%、99.55%，，CD34、CD45阳性率分别为1.40%、1.80%；CFSE结果显示ADSCs压力组细胞的增殖活性较单纯ADSCs培养组显著提高；软骨特异性指标SOX-9，Ⅱ型胶原及aggrecan的基因表达水平在共培养联合压力组中增加最为显著，其中Ⅱ型胶原基因表达水平变化与其蛋白表达量变化相一致。 【结论】动态压应力能促进ADSCs的增殖，并且在联合NPCs共培养微环境下可诱导ADSCs向髓核样细胞分化。
[Abstract]:[objective] to investigate the effects of dynamic compressive stress and co-culture microenvironment on the proliferation and differentiation of adipose mesenchymal stem cells (ADSCs) into nucleus pulposus like cells. [methods] Primary SD rat ADSCs was cultured and amplified in vitro. After identification of cell surface markers by flow cytometry, the third generation of ADSCs was stimulated by controllable cell dynamic compressive stress loading device (90 mm Hg + 12 h / d), and negative group was set up. Cell proliferation activity was measured by CFSE staining. The fluorescence attenuation was detected by flow cytometry and inverted fluorescence microscope was used to observe the fluorescence attenuation at 24 hours after dyeing for 48 h and 72 h for 96 h. At the same time, the alginate microspheres coated with (NPCs) of SD rat nucleus pulposus were co-cultured with ADSCs of the third generation. According to the cell co-culture and compressive stress stimulation or not, they were divided into ADSCs compression group and NPCs culture group. Co-culture group and co-culture combined pressure group, The expression of SOX-9, type 鈪

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