| 等轴牵张应变对骨间充质干细胞成软骨分化早期的影响 |
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Received:June 20, 2018
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| 期刊信息：《中国骨伤》2018年31卷，第9期，第846-852页 |
| DOI：10.3969/j.issn.1003-0034.2018.09.013 |
| 基金项目:国家自然科学基金项目（编号：30973048）；2015年山西省基础研究计划项目（编号：2015011111）；山西省人才专项（编号：201705D211022） |
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目的：观察平面诱导培养条件下，等轴周期性牵张应变刺激对小鼠BMSCs成软骨分化早期的影响，探讨等轴周期性牵张应变刺激在软骨分化过程中早期的作用机制。
方法：选取4周龄KM小鼠16只，雌雄不限，平均体重19.5 g （17~21 g），提取骨髓间充质干细胞，体外培养至第3代，种植于BioFlex细胞培养板，根据实验设计分6组：空白组，普通培养液培养8 d，不给予等轴周期性牵张应变刺激；对照组，成软骨诱导分化培养液培养8 d，不给予等轴周期性牵张应变刺激；实验组，实验组又分4组，均使用成软骨诱导分化培养液培养8 d，期间分别给与1、3、5、7 d等轴周期性牵张应变刺激。于培养第8天收集各组细胞，采用RT-PCR分析SOX9、Col-Ⅱ及ROCK1 mRNA的相对表达量，采用CCK-8法对各组细胞行增殖率检测，使用糖胺聚糖Elisa试剂盒检测上清液糖胺聚糖含量，行番红O及阿利新蓝染色观察细胞外基质（ECM）变化，正态计量资料采用均数±标准偏差表示，对照组与空白组比较采用配对样本t检验，实验组与对照组比较采用单因素方差分析。
结果：（1）培养8 d后，对比对照组，实验组中随加载时间延长，SOX9、Col-Ⅱ mRNA相对表达量呈逐渐增高趋势（P<0.05），而ROCK1 mRNA相对表达量呈下降趋势（P<0.05）；对比空白组，实验组及对照组的ROCK1 mRNA相对表达量均增加（P<0.05）。（2）随加载时间延长，实验组出现先降后升趋势，但是对比空白组及对照组均增高，对照组较空白组明显下降。（3）通过对最后1次换掉的培养基做糖胺聚糖浓度的Elisa检测，实验组内糖胺多糖的分泌量逐渐增加，加载7 d组含量变化较其他组差异有统计学意义（P<0.05）；与空白组比较，实验组及对照组糖胺多糖的分泌量明显增加（P<0.05）。（4）番红O及阿尔新兰染色显示，实验组有成软骨分化趋势，形状随时间加载均逐渐变长，均较对照组明显；实验组内PCM、Col-Ⅱ、GAG含量随力学刺激天数增加逐渐增多均较对照组明显。
结论：平面诱导培养条件下，在BMSCs成软骨分化早期，等轴周期性牵张应变刺激可以促进骨髓间充质干细胞增殖以及向软骨细胞分化，等轴周期性牵张应变刺激可能是通过抑制Rho/ROCK1信号通路来促进成软骨分化。 |
| [关键词]:生物力学 细胞增殖 成软骨分化 |
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| Effect of equiaxial tensile strain in early differentiation of mesenchymal stem cells into cartilage cells |
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Abstract:
Objective: To observe the effect of cyclic equiaxial tensile strain in the early differentiation of bone marrow mesenchymal stem cells(BMSCs) into cartilage in mouse under conditions of two-dimensional culture,and to investigate the mechanism of cyclic equiaxial tensile strain in early chondrogenic differentiation.
Methods: Sixteen KM mouse aged 4 weeks were selected,male and female unlimited,with an average weight of 19.5 g (17 to 21 g). After extracting and isolating the BMSCs from KM mouse,then subculture the BMSCs to the 3rd generation. Seed the cells in the biological plate(BioFlex). According to experimental design,the cells were divided into 6 groups,blank group:ordinary culture medium was cultured for 8 days without isometric cyclic tensile strain stimulation. Control group:chondrogenic differentiation medium was used to culture for 8 days without isometric cyclic tensile strain stimulation. Experimental group:the experimental group was divided into 4 groups,all of which were cultured with chondrogenic differentiation medium for 8 days. During which isometric cyclic tensile strain stimulation was given for 1,3,5 and 7 days respectively. At the 8th day,all the cells were collected,the expression of the Sox9,Col-Ⅱand ROCK 1 signaling pathway-related molecules was analyzed by RT-PCR. Cells in each group were extracted,and the efficiency of cell proliferation in each group was detected by CCK-8. Glycosaminoglycan content in medium changed last was detected using ELISA. Pericellular matrix was observed by Safranin O staining and Alcian Blue staining. Normal measurement data using mean±standard deviation compared between the blank group and control group using paired t-test,compared between the experimental group and relative group using single factor analysis of variance.
Results: (1) After 8 days of culture,compared with the control group,the relative expression of Sox 9 and Col-Ⅱ mRNA in the experimental group increased gradually with the increase of loading time(P<0.05),while the relative expression of ROCK1 mRNA decreased(P<0.05). Compared with the blank group,the relative expression of ROCK1 mRNA in experimental group and control group increased (P<0.05). (2)With the increase of loading time,the experimental group showed a trend of decreasing at first and then increasing,but compared with the blank group and the control group,the control group decreased significantly. (3)Glycosaminoglycan content in the medium changed last was detected by ELISA. The glycosaminoglycans in the experimental group increased gradually,and the content changes on 7 days loading group were statistically significant compared with other groups(P<0.05). (4)Safranin O and Alcilan staining showed that there was a tendency of cartilage differentiation in the experimental group,and the shape gradually increased with time,which was more obvious than that in the control group; The PCM,Col-Ⅱ and GAG in the experimental group increased gradually with the increase of mechanical stimulation days,which were more obvious than those in the control group.
Conclusion: Under conditions of two-dimensional culture,in the early differentiation of mesenchymal stem cells into cartilage,cyclic equiaxial tensile strain can promote the proliferation of BMSCs and the differentiation into chondrocytes. Moreover,cyclic equiaxial tensile strain may promote chondrogenic differentiation through inhibiting the Rho/ROCK 1 signaling pathway. |
| KEYWORDS:Biomechanics Cell proliferation Chondrogenic differentiation |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 杨自权,门亚勋.等轴牵张应变对骨间充质干细胞成软骨分化早期的影响[J].中国骨伤,2018,31(9):846~852 |
| 英文格式: | YANG Zi-quan,MEN Ya-xun.Effect of equiaxial tensile strain in early differentiation of mesenchymal stem cells into cartilage cells[J].zhongguo gu shang / China J Orthop Trauma ,2018,31(9):846~852 |
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