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活性维生素D3对白介素-6作用下肌原细胞增殖和分化的影响及机制研究
1,25(OH)2-vitamin D3 prevents interleukin-6 induced muscle atrophy by stimulating differentiation and inhibiting myostatin expression in C2C12 muscle cells
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DOI:
作者:
王会玲
Wang Huiling, Li Yan, Xu Kai, Liu Nanmei, TianJjun, Zhang Jinyuan
作者单位:
上海455医院
Department of Nephrology, 455th Hospital of PLA, The Institute of Nephrology of Nanjing Military Command, Shanghai 200052, China
关键词:
蛋白质-能量消耗;慢性肾脏病;C2C12成肌细胞;维生素D;白介素-6
protein-energy wasting; chronic kidney disease; C2C12 muscle cells; Vitamin D; interleukin-6
摘要:
目的 探讨1,2A5(OH)活性vitD3(1,25-D3)对白介素-6(interleukin-6, IL-6)刺激下成肌细胞增殖和分化的影响及机制。方法 体外培养小鼠C2C12肌原细胞株,2%马血清使细胞分化为肌索,给予IL-6(0~100ng/ml)和1,25-D3刺激细胞,光镜下观察细胞增殖和肌管形态;Western印迹法检测vitD3 受体(VDR)、成肌调节分子MyoD、 Myogenin和肌动蛋白重链(MHC)等蛋白表达水平,荧光定量PCR检测myostatin、Atrogin-1、MyoD、Myogenin mRNA转录水平。结果 光镜下观察C2C12细胞呈梭形生长,72h后达80%培养皿,2%马血清培养72h后,细胞可分化为条索状肌索。IL-6(20ng/mL)可引起细胞增殖缓慢,其中100ng/ml组细胞见较多坏死脱落,分化后的肌索较纤细稀疏,直径呈不同程度下降。以1,25-D3(100nM)处理后,可见细胞增殖密度较对照略降低,但分化后的肌索明显粗壮,肌索直径显著增加(p<0.01)。western blot结果显示1,25-D3作用后VDR表达显著上调;IL-6对VDR表达无影响,但可使myoD、myogenine表达水平下调,使MHC蛋白表达水平降低约45%。IL-6作用后对VDR表达无影响,但可使生肌调节因子myoD、myogenin表达水平下调,使MHC下调约45%。1,25-D3可改善IL-6引起的生肌调节因子表达下调,并使MHC上调约56%。荧光定量PCR结果显示IL-6使肌索myostatin mRNA转录水平上调2.79倍,但Atrogin-1仅轻度上调,同时使myogenine、myoD mRNA下调;1,25-D3可抑制IL-6引起的myostatin mRNA表达上调,并使MyoD、Myogenin mRNA表达上调。 结论 1,25-D3可使肌细胞VDR表达显著上调,促进肌细胞分化,使肌纤维直径增粗肥大,抑制炎症因子IL-6作用下的肌萎缩;该作用与抑制myostatin mRNA转录,并促进成肌分化分子MyoD、Myogenin表达有关。
Objective To investigate the effect and mechanism of 1,25-dihydroxy-vitamin D (1,25-D3) on proliferation and differentiation of C2C12 myoblast cells exposure with interleukin-6(IL-6). Methods Cultured mouse C2C12 cells,2% horse serum induced cells differentiated into myotubes. After treated with IL-6( 0~100ng/ml) or 1,25-D3(100nM), the cells proliferation and myotube’s morphology was observed by light microscopy, the protein expression of vitamin D receptor(VDR) and myogenic regulatory factors such as MyoD and Myogenin and actin heavy chain (MHC) were measured by Western blotting. The mRNA transcription levels of muscle atrophy molecular such as myostatin and Atrogin-1, the mRNA of MyoD and Myogenin were quantitative by real time-PCR. Results The growth myoblasts were like fusiform, proliferation to 80% dish after 72h. After changed medium with 2% horse serum, the cells differentiate into myotube. When chronic exposure to IL-6(>20ng/ml) , the cells proliferated slowly, and more necrotic cells was observed when with IL-6 (100ng/ml); the size of myotubes were slim, and the diameter was decreased markedly. After treated with 1,25-D3 (100nM) , cells density were slightly lower than the control, but myotube seems clearly strong, and the diameter increased significantly (p <0.01). 1,25-D3 significantly induced VDR expression. IL-6 had no effluence on VDR expression, but induced the expression of myoD and myogenin down-regulation, and caused MHC protein expression decreased about 45%. 1,25-D3 improved the IL-6-induced myogenic regulatory factor down-regulation, increased MHC expression about 56%. Quantitative PCR showed that IL-6 enhanced myostatin mRNA transcription but had little influence in Atrogin-1, while myogenin and myoD mRNA down-regulated; 1,25-D3 inhibited the mRNA up-regulation of myostatin induced by IL-6; and up-regulated MyoD and Myogenin mRNA transcriptional levels. Conclusion 1,25-dihydroxyvitamin D active VDR, promoted muscle cells differentiation, caused myotubes hypertrophy. 1,25-D3 prevents muscle atrophy induced by inflammatory cytokine interleukin-6. The mechanism might related to 1,25-D3 promote myogenic differentiation, up-regulate myoD and myogenin expression, and inhibit the myostatin transcription.
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