肌肉干细胞条件培养基对皮肤成纤维细胞功能的影响

Effects of muscle stem cell conditioned medium on the proliferation, migration and differentiation of fibroblasts in vitro

[目的]探讨肌肉干细胞条件培养基(muscle stem cell conditioned medium, MuSC-CM)对小鼠皮肤成纤维细胞的增殖迁移及向肌成纤维细胞分化功能的影响。[方法]获取新西兰大白兔后肢比目鱼肌,采用差速贴壁培养法分离培养肌肉干细胞,制备肌肉干细胞条件培养基并用超滤离心管浓缩。将成纤维细胞分为对照组(即不添加条件培养基组),5倍浓度MuSC-CM组,10倍浓度MuSC-CM组和20倍浓度MuSC-CM组。通过cck8实验检测不同浓度的MuSC-CM对成纤维细胞增殖作用的影响,通过细胞划痕实验检测10倍浓度的MuSC-CM对成纤维细胞迁移的影响,通过Western Blot和3D胶原回缩实验检测MuSC-CM对成纤维细胞向肌成纤维细胞分化功能的影响。[结果]cck8实验结果示,不同浓度实验组中成纤维细胞的增殖情况均显著高于对照组,且在48 h和72 h处呈浓度依赖性趋势。细胞划痕实验中,10倍浓度MuSC-CM处理的成纤维细胞在24、48 h的迁移率均高于对照组,差异有统计学意义。Western blot结果示,不同浓度实验组中肌成纤维细胞α-SMA,蛋白表达量显著高于对照组,3D胶原回缩实验结果示,24 h后不同浓度实验组的3D胶原回缩程度显著高于对照组,差异均有统计学意义。[结论]本实验证明MuSC-CM能加快成纤维细胞的增殖与迁移,同时促进成纤维细胞向肌成纤维细胞分化。

[Objective] To investigate the efforts of muscle stem cell conditioned medium （MuSC-CM） on the proliferation, migration and differentiationof fibroblasts in vitro. [Methods] Muscle stem cells were isolated from the soleus of New Zealand white rabbit according to the different time taken for the cells to adhere. MuSC-CM was prepared and concentrated by ultrafil- tration centrifuge tube. Fibroblasts were divided into several groups, including control group which did not add any conditioned medium, 5-fold concentration MuSC-CM group, 10-fold concentration MuSC-CM group and 20-fold concentration MuSC-CM group. CCK8 assay was performed to study the efforts of different concentrations of MuSC-CM on fibroblasts prolifer- ation, the effort of MuSC-CM on migration of fibroblasts was determined by scratch assay and the efforts of MuSC-CM on fibro- blasts differentiation was detected by Western Blot and 3D Collagen gel contraction assay. [Results] The MuSCs were isolated from soleus muscle with Desmin detected. CCK-8 assay revealed that MuSC-CM increased proliferation of fibroblasts in a dose- independent manner. In term of the scratch assay, the fibroblasts migration rate at 24 h and 48h in 10-fold MuSC-CM group was significantly higher than that in control group. In addition, Western blot showed that the fibroblasts treated with MuSC-CM had a significant higher expression of α- SMA than the control. Moreover, the results of 3D Collagen gel contraction assay showed that MuSC-CM can accelerate the contraction of 3D Collagen gel. [Conclusion] The MuSC-CM does increase the proliferation and migration of fibroblasts, as well as promote the differentiation of fibroblasts into myofibroblasts.