肌卫星细胞功能异常在慢性肾脏病骨骼肌消耗中的机制

Chronic kidney disease caused muscle satellite cell dysfunction induced muscle atrophy

目的:骨骼肌消耗是慢性肾脏病(CKD)的主要特征,肌卫星细胞(muscle satellite cells,MSC)是具有自我增殖和更新能力的成肌干细胞,MSC功能异常可引起肌肉萎缩。本研究探讨MSC在CKD小鼠骨骼肌消耗中的作用机制。方法:采用5/6肾切除制作小鼠CKD模型,以假手术组为对照组。以免疫组化标记胫骨前肌层粘连蛋白(laminin),测量肌纤维横截面积,并计算面积百分位图;RT-PCR检测腓肠肌Pax-7、MyoD、Myf-5、Myogenin及myostatin mRNA表达水平;分离并原代培养骨骼肌MSC,原位免疫组化检测Pax-7和MyoD表达,检测分化后新形成的肌纤维胚胎型肌球蛋白重链(eMyHC)。结果:造模一个月后,CKD小鼠体重和骨骼肌重量明显下降,形态学表现为胫骨前肌明显变细,肌纤维横截面积减少,面积百分位图明显左移;肌肉组织的Pax-7、MyoD、Myf-5和Myogenin mRNA表达水平均有不同程度下降,myostatin mRNA表达则明显上调;原代培养的MSC中,CKD组的Pax-7和MyoD阳性细胞数目明显低于对照组,分化后新形成的eMyHC阳性肌纤维数目也明显降低。结论:CKD可引起显著的骨骼肌萎缩,使MSC增殖和分化功能下降,myostatin水平升高可能是抑制肌卫星细胞功能的重要原因。

Objective：Muscle wasting is a serious complication associated with excessive morbidity and mortality in chronic kidney disease （CKD）. Muscle satellite cells （MSC） are myogenic precursors responsible for skeletal muscle regeneration. Reduced MSC function contributes to atrophy. In this study, the mechanism of MSC in CKD caused muscle atrophy was investigated. Methodology：Mouse CKD model was made by subtotal nephrectomy, and the Sham was as control. The muscle size of tibia anterior （TA） was measured under microscope after immunohistochemistry staining the Laminin and calculated with software. The mRNA expression of Pax-7, MyoD, Myf-5, Myogenin and myostatin mRNA was measured by RT-PCR. MSC was isolated and cultured from gastrocnemius and then immunostained to detect the expression of MyoD and Pax-7. The eMyHC was alao immunostained to identify the new fibers. Results ： The bodyweight and TA muscle weight in CKD mouse were decreased compared with Control. The cross-sectional area of muscle fibers was significantly smaller than that in control, and the shrinkages of fibers sizes caused leftward shift in fiber size distribution. The mRNA of myogenic markers（ Pax-7 ,MyoD,Myf-5 ,myogenin） were reduced, but the myostatin mRNA were increased significantly in the gastrocnemius of CKD mouse. The MyoD and Pax-7-positive cell numbers isolated from muscles of CKD was decreased, and the eMyHC-positive fibers was decreased too. Conclusion： CKD impaired MSC proliferation and differentiation, and delayed MSC differentiation into myotubes which induced skeletal muscle atrophy. The myostatin might be a key regulator caused dysfunction of MSC.