中文摘要：
目的:成肌细胞是起源于肌肉卫星细胞的肌源细胞,具有分化、增殖等能力,成肌细胞衰老与骨骼肌减少相关疾病的发病密切相关。自噬作为细胞生存的一种应激反应,在细胞自稳中发挥重要的作用,有研究表明随着衰老发生,自噬活性逐渐被抑制。而自噬对成肌细胞衰老的调控作用及调控机制尚不十分明确。本研究使用D-半乳糖诱导C2C12细胞模拟骨骼肌成肌细胞衰老,以研究自噬与衰老成肌细胞的作用关系及机制,为骨骼肌减少相关疾病的诊疗提供新的参考。方法:1.D-半乳糖干预C2C12成肌细胞后,通过β-半乳糖苷酶染色、Western-blot、JC-1线粒体膜电位、超氧化物歧化酶实验对β-半乳糖苷酶的活性、P53和P16蛋白表达水平、线粒体功能和细胞抗氧化应激活性等衰老相关指标进行检测。2.D-半乳糖干预C2C12成肌细胞后,通过western-blot分析P62、LC3蛋白表达,免疫荧光检测P62的变化,观察自噬水平改变。3.实验分为四组:对照组(MOCK组)、D-半乳糖组(D-GAL组)、雷帕霉素预处理的D-半乳糖组(D-GAL+RAPA组)、3-甲基腺嘌呤预处理的D-半乳糖组(DGAL+3-MA组)。β-半乳糖苷酶染色、线粒体膜电位JC-1、超氧化物歧化酶和Westernblot实验检测C2C12成肌细胞衰老水平变化。EdU-488和流式细胞术分析细胞增殖水平与周期分布改变。4.实验分为四组:对照组(MOCK组)、D-半乳糖组(D-GAL组)、雷帕霉素预处理的D-半乳糖组(D-GAL+RAPA组)、3-甲基腺嘌呤预处理的D-半乳糖组(DGAL+3-MA组)。用Western-blot检测Hippo信号通路相关蛋白(Mst1/2、p-LATs1/2、YAP、p-YAP1)的表达变化。结果:1.D-半乳糖诱导C2C12成肌细胞衰老:D-半乳糖干预后,C2C12细胞表达β-半乳糖苷酶、P53和P16增加;超氧化物歧化酶活性被抑制;线粒体膜电位染色多呈绿色荧光,线粒体损伤增加。2.D-半乳糖抑制自噬活性:免疫印迹结果显示从D-半乳糖干预后9小时起,微管相关蛋白1轻链3由Ⅰ型向Ⅱ型转化减少,自噬相关蛋白P62增多;P62免疫荧光观察到颗粒状荧光在胞浆聚集。3.自噬抑制D-半乳糖诱导的C2C12成肌细胞衰老:与D-半乳糖干预组相比,雷帕霉素预处理细胞后,衰老相关的半乳糖苷酶、P53和P16蛋白减少,超氧化物歧化酶活性增强,线粒体膜电位红色荧光增多。4.自噬抑制D-半乳糖对C2C12成肌细胞增殖、周期的影响:与D-半乳糖干预组相比,雷帕霉素预处理细胞后,EdU阳性的增殖细胞增多,处于G0/G1期细胞减少。5.自噬通过抑制Hippo信号通路抑制C2C12成肌细胞衰老:免疫印迹结果显示D-半乳糖促进Hippo信号通路相关蛋白LATS1/2、YAP1磷酸化,雷帕霉素预处理后,磷酸化的LATS1/2、YAP1蛋白减低。结论:D-半乳糖诱导C2C12成肌细胞衰老,抑制成肌细胞自噬活性和增殖能力,诱导细胞周期阻滞。增强自噬水平可以通过抑制Hippo信号通路抑制C2C12成肌细胞衰老。这些结果为研究肌源细胞衰老的发生发展提供理论基础,为骨骼肌减少相关疾病的防治提供新的思路。
英文摘要：
Objective:Myoblast is a myogenic cell derived from muscle satellite cells,which has the ability of differentiation,proliferation,and etc.Myoblast senescence was the causes of skeletal muscle atrophy and sarcopenia.As a mechanism of cell survival,autophagy played a pivotal role in maintaining the homeostasis of the cell environment.Some studies have shown that autophagy activity is gradually inhibited as aging occurs.However,the regulatory effect and mechanism of autophagy on myoblast senescence has not been clear.In this study,C2C12 myoblasts induced by d-galactose were used to establish a skeletal muscle cell aging model in order to study the effect of autophagy on C2C12 myoblasts aging and to provide new reference for diagnosis and treatment of skeletal muscle reduction-related diseases.Methods:1.The effect of d-galactose on C2C12 myoblasts was studied.β-galactosidase staining,western blot,superoxide dismutase assay and mitochondrial membrane potential assay was used to detect the expression of aging-related β-galactosidase,P53 and P16,the antioxidant stress activity and mitochondrial function.2.After D-galactose intervened C2C12 myoblasts,the expression of autophagy-related protein P62 and LC3 was tested by western-blot,and the changes of P62 were detected by immunofluorescence assay.3.The experiment was divided into four groups: control group(MOCK group),Dgalactose group(D-GAL group),rapamycin pretreatment d-galactose group(D-GAL+RAPA group)and 3-methylpurine pretreatment d-galactose group(D-GAL+3-MA group).β-galactosidase staining,mitochondrial membrane potential,superoxide dismutase and Western-blot assay were used to detect the changes of C2C12 myoblasts in aging level.EdU-488 was devoted to analyze cell proliferation and flow cytometry was used to test the distribution of cell cycle.4.The experiment was divided into four groups: control group(MOCK group),Dgalactose group(D-GAL group),rapamycin pretreatment d-galactose group(D-GAL+RAPA group)and 3-methylpurine pretreatment d-galactose group(D-GAL+3-MA group).Westernblot was used to detect the expressions of Hippo signaling pathway related proteins(pLATs1/2,Mst1/2,YAP,p-YAP1).Results:1.D-galactose induced C2C12 myoblast senescence: After d-galactose treatment,the number of C2C12 myoblast aging-related β-galactosidase staining positive cells increased,The aging-related proteins P53 and P16 increased.Superoxide dismutase activity was degraded.Mitochondrial membrane potential staining showed increasing green-fluorescence.2.D-galactose inhibited autophagy activity: Western blotting results showed that after 9 hours of d-galactose treatment,the conversion of LC3-I to LC3-II is reduced,and degradation of autophagy associated protein P62(The 62 kDa sequestosome 1 protein,P62 protein)is inhibited;P62 granular fluorescence aggregation was observed under fluorescence microscope.3.Autophagy inhibited C2C12 myoblasts senescence induced by d-galactose: Compared with D-GAL group,in D-GAL+RAPA group,the activity of β-galactosidase and the expression of aging-related proteins P53 and P16 were decreased,concomitant with the increases red-fluorescence of mitochondrial membrane potential and superoxide dismutase.4.Autophagy inhibited the effects of d-galactose on proliferation and cycle of C2C12 myoblasts: Compared with D-GAL group,in D-GAL+RAPA group,the number of EdU positive cells increased and G0/G1 phase cells decreased.5.Autophagy inhibited C2C12 myoblast senescence by blocking Hippo signaling pathway: Western blot results show that D-galactose promotes phosphorylation of Hippo signaling pathway related proteins LATS1/2 and YAP1,and phosphorylated proteins LATS1/2 and YAP1 decrease after rapamycin pretreatment.Conclusion:D-galactose induced C2C12 myoblast senescence,inhibited autophagy activity and proliferation,and arrest cell cycle.Enhancing autophagy activity can inhibit C2C12 myoblast senescence by regulating Hippo signaling pathway.These results may provide a theoretical basis for us to understand the occurrence and development of myogenic cell senescence,and provide new ideas for the prevention and cure of skeletal muscle reductionrelated diseases.
