机械力对小鼠成纤维细胞Ⅰ、Ⅲ型胶原蛋白表达的影响研究

目的:探讨机械力对小鼠成纤维细胞L929细胞Ⅰ、Ⅲ型胶原蛋白(CollagenⅠ、CollagenⅢ)表达的影响及其可能机制。方法:选取L929细胞为研究对象,使用四点弯曲细胞力学加载系统对细胞进行机械力干预,加力时间为4小时,加力频率为1 Hz;L929细胞分别经0、1333μ和5333μ机械力干预后,采用CCK8检测不同大小机械力对细胞活性的影响。L929细胞分别经0和5333μ机械力干预后,采用Western blot检测两组细胞转化生长因子β1(TGF-β1)、Smad蛋白(Smad2、Smad3、p-Smad2、p-Smad3)和CollagenⅠ、CollagenⅢ的表达,采用实时荧光定量PCR检测两组细胞TGF-β1、Smad2、Smad3、CollagenⅠ和CollagenⅢmRNA的表达,采用细胞免疫荧光检测细胞内Smad2/3的核转移情况。结果:与对照组相比,L929细胞经1333μ机械力干预后细胞增殖活性明显上升,而5333μ机械力干预后细胞增殖活性显著下降(P0.05)。与对照组相比,5333μ组细胞TGF-β1、pSmad3、CollagenⅠ、CollagenⅢ水平及TGF-β1、CollagenⅠ、CollagenⅢmRNA表达均明显降低(P0.05),且Smad2/3核转移被明显抑制;而细胞Smad2、Smad3、p-Smad2蛋白及Smad2、Smad3 mRNA表达差异均无统计学意义(P0.05)。结论:5333μ机械力可降低小鼠成纤维细胞CollagenⅠ、CollagenⅢ的表达,其机制可能与TGF-β1/Smads信号转导通路抑制有关。

Mechanical Stress Regulates the Expressions of Collagen Ⅰ and Collagen Ⅲ in Mouse Fibroblasts

Objective:To investigate the effect of mechanical stress on the expression of collagen Ⅰ and Ⅲ in mouse fibroblast(L929) cells and to reveal the possible molecular mechanism.Methods:We chose L929 cells in the experimental study.Cells were stretched by four-point bending system for 0,1333,5333μstrain for the frequency of 1.0 Hz and 4 hours.The viability of L929 cells was evaluated using Cell Counting Kit-8 assay.After cells were stretched for 0 and 5333 μstrain,the protein expression levels of TGF-β1,Smad2,Smad3,p-Smad2,pSmad3,Collagen Ⅰ and Ⅲ were measured using Western blot analysis.Real-time Quantitative PCR was used to detected the mRNA expression levels of TGF-β1,Smad2,Smad3,Collagen Ⅰ and Collagen Ⅲ.The nuclear translocation of Smad2/3 was investigated by immunocytofluorescent.Results:The viability of cells treated with 1333 μstrain mechanical stress was increased than that of the control group,while the group of 5333 μstrain was significantly decreased than that of the control group(P ＜0.05);Compared with the control group,the protein expression levels of TGF-β1,p-Smad3,Collagen Ⅰ,Collagen Ⅲ and the RNA expression levels of TGF-β1,Collagen Ⅰ,Collagen Ⅲ were remarkably declined in group of 5333 μstrain group (P ＜ 0.05).The nuclear translocation of Smad2/3 was inhibited in 5333 μstrain group.But there was no significant differences of the protein expressions of Smad2,Smad3,p-Smad2 and the RNA expressions of Smad2,Smad3 between the two groups(P＞0.05).Conclusions:5333 μ mechanical stress could reduce the expression of Collagen Ⅰ and Ⅲ in mouse fibroblast cells.The mechanism may be related to inhibition of TGF-β1/Smads signaling transduction pathway.