TGF-β/Smad信号通路在Follistatin调节鸭骨骼肌卫星细胞增殖过程中的作用机制

【目的】卵泡抑素（Follistatin）能够调节骨骼肌肥大和脂肪沉积，可促进骨骼肌卫星细胞增殖。拟采用体外重组 Follistatin处理增殖期的鸭骨骼肌卫星细胞，阐明TGF-β/Smad信号通路在Follistatin调节鸭骨骼肌卫星细胞增殖过程中的作用机制。【方法】以孵化14 d的鸭胚为试验材料，采用差速贴壁的方法分离骨骼肌卫星细胞，待细胞长到70%—80%时，将培养基换成含有浓度分别为0、1、10、100 ng·mL^-1的Follistatin培养基，继续培养36 h后，采用CCK-8检测骨骼肌卫星细胞增殖情况；使用抗pax7抗体染色，DAPI染核，鉴定骨骼肌卫星细胞；采用real-time qPCR方法检测Follistatin对骨骼肌卫星细胞增殖过程中的标记基因PCNA、生肌因子基因MyoD和TGF-β信号通路中TGF-β、Smad2和Smad3的表达的影响。【结果】在倒置显微镜下观察，传代培养12 h鸭骨骼肌细胞一部分未贴壁呈圆形，一部分贴壁呈梭形。24 h后细胞全部贴壁，细胞略有变长。2 d后细胞继续增多，且呈长梭形。3 d后细胞数目增加，个别细胞融合。4 d后细胞数目进一步增加，细胞变粗，个别细胞融合。5 d后有少量细胞开始分化，细胞进一步融合。Pax7免疫荧光染色分析显示，95%以上的细胞中Pax7呈阳性表达；CCK-8检测细胞增殖分析表明，不同浓度的Follistatin处理鸭骨骼肌卫星细胞后，各处理组细胞增殖均显著高于对照组(P＜0.01)，且10 ng·mL^-1 Follistatin处理鸭骨骼肌卫星细胞增殖效果最明显，为最佳处理浓度；与对照组相比，10 ng·mL^-1 Follistatin处理组的MyoD基因表达量显著下降(P＜0.05)，PCNA基因表达量显著升高(P＜0.05)，Myf5基因表达量显著升高，TGF-β和Smad2基因表达显著升高(P＜0.05)，且Smad3基因表达量极限著升高(P＜0.01)；Western blotting检测蛋白表达水平结果表明，与对照组相比，TGF-β、Smad2 和Smad3磷酸化水平也显著升高。【结论】10 ng·mL^-1 Follistatin能显著促进鸭骨骼肌卫星细胞增殖，这一过程可通过TGF-β/Smad信号通路实现。使用最佳Follistatin处理浓度能够显著促进鸭骨骼肌卫星细胞增殖,该研究为鸭骨骼肌生长发育调控机理研究奠定分子基础。

The Regulated Mechanism of Follistatin on the Proliferation Process of Duck Skeletal Muscle Satellite Cell Involved in TGF-β / Smad Signaling Pathway

【Objective】Follistatin can regulate skeletal muscle hypertrophy and fat deposition, and promote proliferation of the skeletal muscle satellite cell. The study intends to use in vitro recombination follistatin to treat the duck skeletal muscle satellite cell, which can be proved that the functional mechanism of TGF-β/smad signaling pathway is playing a role in its proliferation process.【Method】 Based on differential centrifugation technology, skeletal muscle satellite cells isolated from embryonic 14-day ducks were treated by proliferation medium containing 0,1,10 and 100 ng·mL^-1 follistatin, respectively, after the cells density reached 70%-80% confluence. After incubation for 36 h, the degree of skeletal muscle satellite cells proliferation were tested by CCK-8. The cells were identified by immunofluorescence staining with pax7 antibody. Real-time qPCR was performed to measure the genes of expression differntiation including proliferation marker gene PCNA, and myogenic MyoD, smad2/3, TGF-βcaused by follistatin supplement.【Result】Under the inverted microscope, ti was found that a part of duck skeletal muscle cells did not adhere to round, a of cells part of cells adhered to fusiform 12 hours of culture. Adherent cell number increased and cells slightly longer after 24 h. Cells completely adherent, and fusiform, on day 2. The cell number increased, some cells differentiated, after 3 days. The number of cells further was increased, the cells thicker, some cells started to fuse after 4 days. A small amount of cells began differentiation, and further fusion of the cell on day five. The result of immunofluorescence staining showed that pax7 in more than 95% of the skeletal muscle satellite cells was positive, and the population of cells stained with Pax7 was suggested that the group of 10 ng·mL^-1 follistatin was significantly higher than the control group (P＜0.01). The gene expression analysis showed that compared with the control group, the expression level of MyoD in 10 ng·mL^-1 follistatin treated group was significantly decreased (P＜0.05), whereas, the expression levels of PCNA, TGF-β and Smad2 in follistatin treated groups were significantly increased (P＜0.05). The expression of Smad3 gene ultimately significantly increased (P＜0.01). Results of detection of protein expression by Western blotting showed that compared with the control group, TGF-β protein and Smad2/3 phosphorylat ion protein levels were also significantly increased.【Conclusion】Results of study suggested that follistatin promoted the expression of PCNA gene and reduced MyoD gene expression in duck skeletal muscle satellite cell. Furthermore, follistatin raised the expression of TGF-β, Smad2/3. Generally, 10 ng·mL^-1 concentration of follistatin could significantly promote proliferation of the duck skeletal muscle satellite cell, the same that TGF-β / Smad signaling pathway maybe play a important role in it.