滑膜间充质干细胞成纤维软骨分化条件初步探索

发布时间：2018-10-30 12:22

【摘要】：目的采用正交实验研究滑膜间充质干细胞(synovial derived MSCs,SMSCs)成纤维软骨分化的条件。方法 5只成年新西兰白兔,活检取滑膜组织。贴壁法获取SMSCs后采用流式细胞仪及成脂、成骨、成软骨诱导分化鉴定。根据预实验与文献综述寻找与SMSCs成纤维软骨分化可能相关的条件,采用缺失实验初筛必要条件后,TGF-β1、BMP-2、地塞米松、脯氨酸、柠檬酸(ascorbic acid,ASA)、丙酮酸、胰岛素+转铁蛋白+亚硒酸预混液、牛血清白蛋白、b FGF、间断静水压、BMP-7、IGF纳入正交实验,采用SPSS18.0统计软件设计L60(212)正交实验及表头,定义2水平条件,在SMSCs-三维小肠黏膜下层(small intestinal submucosa,SIS)支架上诱导成纤维软骨分化。使用流式细胞仪计数,检测CD151+/CD44+细胞并记录SMSCs向纤维软骨分化的转换率,采用免疫组织化学染色,结合细胞形态、甲苯胺蓝染色、半定量RT-PCR检测SOX9、聚集蛋白聚糖、Ⅰ型胶原(collagen typeⅠ,ColⅠ)、ColⅡ、ColⅨ基因表达,进一步验证结果。检验指标rate是CD151+/CD44+细胞与高表达ColⅠ细胞的比例乘积。同时采用Pico Green Assay测量细胞总DNA量,以反映细胞扩增情况。正交实验结果采用直观观察和主体间方差分析方法,考虑部分因子间的1阶交互作用,组间差异采用LSD和q检验验证,使用Ⅲ型平方和校正模型,检验水准α=0.05。结果实验获取的细胞为SMSCs,细胞倍增时间为28 h。成纤维软骨分化过程中SMSCs-三维SIS支架体积5 d倍增,14 d后得到甲苯胺蓝染色阳性的细胞-支架复合物。正交实验结果直观分析示,TGF-β1对成纤维软骨分化转化率的作用最明显,BMP-7采用水平2有利于得到更高的转化率,但与BMP-2存在交互作用;其余第1区组水平值加和高于22.5的因子有DEX、ASA、ITS、转铁蛋白、b FGF,模型的相关性好。方差分析校正模型P=0.000,能够满足实验设计;截距P=0.000,说明各因子对因变量影响差异不完全相同。TGF-β1、ASA、b FGF、IGF对因变量调控作用较其他因子显著,差异有统计学意义,与直观观察结果相似。结论 TGF-β1、ASA、b FGF、IGF显著影响SMSCs成纤维软骨分化,通过合理调整上述因子浓度,可显著提高SMSCs成纤维软骨分化转化率;精确的调控条件和调控机制有待进一步探索。
[Abstract]:Objective to study the condition of fibrocartilage differentiation of synovial mesenchymal stem cells (synovial derived MSCs,SMSCs) by orthogonal experiment. Methods 5 adult New Zealand white rabbits were biopsied for synovial tissue. SMSCs obtained by adherent method was identified by flow cytometry, adipogenesis, osteogenesis and cartilage-induced differentiation. According to the pre-experiment and literature review, the possible conditions related to the differentiation of SMSCs fibroblast cartilage were found. After screening the necessary conditions for TGF- 尾 _ 1 BMP-2, dexamethasone, proline, citric acid (ascorbic acid,ASA), pyruvate. Insulin transferrin selenite premixed solution, bovine serum albumin (BSA), b FGF, intermittent hydrostatic pressure, BMP-7,IGF was included in orthogonal experiment, L60 (212) orthogonal experiment and its head were designed by SPSS18.0 software, and 2 level conditions were defined. Fibrocartilage differentiation was induced on SMSCs- 3 D small intestinal submucosal (small intestinal submucosa,SIS scaffold. Flow cytometry was used to detect CD151 / CD44 cells and to record the conversion rate of SMSCs into fibrochondrocytes. Immunohistochemical staining, cell morphology, toluidine blue staining and semi-quantitative RT-PCR were used to detect SOX9, aggregation proteoglycan. The expression of (collagen type 鈪，

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