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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。
Objective To study the conditions of fibroblastic differentiation of synovial derived mesenchymal stem cells (synovial derived MSCs, SMSCs) by orthogonal experiment. Methods Five adult New Zealand white rabbits were biopsied for synovial tissue. SMSCs were obtained by adherence method and then differentiated by flow cytometry and adipogenic, osteogenic, and chondrogenic induction. According to the pre-experiment and literature review, we found the possible conditions related to the fibroblastic chondrogenic differentiation of SMSCs. After the necessary conditions for screening of TGF-β1, BMP-2, dexamethasone, proline, ascorbic acid ), Pyruvate, insulin + transferrin + selenite premix, bovine serum albumin, b FGF, intermittent hydrostatic pressure, BMP-7 and IGF were included in the orthogonal experiment. ) Orthogonal experiments and headers were used to define the 2-level conditions and induce fibroblastic cartilage differentiation on SMSCs-small intestinal submucosa (SIS) scaffolds. Using flow cytometry, CD151 + / CD44 + cells were detected and the conversion rate of SMSCs to fibrocartilage differentiation was recorded. Immunohistochemical staining was used to detect the expression of SOX9, aggrecan , Collagen typeⅠ (ColⅠ), ColⅡ, ColⅨ gene expression, to further verify the results. The test indicator rate is the ratio product of CD151 + / CD44 + cells to highly expressed Col I cells. Pico Green Assay was also used to measure total cellular DNA to reflect cell expansion. Orthogonal experimental results using the method of intuitive observation and analysis of variance between subjects, taking into account the first-order interaction between some factors, the differences between groups using LSD and q test validation, the use of type Ⅲ sum of squares correction model, the test level α = 0.05. Results The cells obtained in the experiment were SMSCs with doubling time of 28 h. The size of SMSCs-3D SIS scaffolds was doubled during fibroblastic differentiation, and the cell-scaffold complexes with toluidine blue staining were obtained after 14 days. The results of orthogonal experiment showed that the effect of TGF-β1 on fibroblastic cartilage differentiation was the most obvious. BMP-7 at level 2 was conducive to higher conversion rate but had interaction with BMP-2; There was a good correlation between DEX, ASA, ITS, transferrin and bFGF in models with the sum of regional blockade values higher than 22.5. Analysis of variance model P = 0.000, to meet the experimental design; Intercept P = 0.000, indicating that the various factors on the dependent variable differences are not exactly the same. The effects of TGF-β1, ASA, b FGF and IGF on the dependent variables were significantly different from other factors, the difference was statistically significant, which was similar to the visual observation. Conclusion TGF-β1, ASA, b FGF and IGF significantly affect the fibroblastic cartilage differentiation of SMSCs. By adjusting the concentration of these factors reasonably, the conversion rate of fibroblast cartilage into SMSCs can be significantly increased. The precise regulatory conditions and regulatory mechanisms need to be further explored.