1,25羟活性维生素D3对白细胞介素-6作用下肌原细胞增殖和分化的影响  

1,25(OH)_2-vitamin D3 prevents interleukin-6 induced muscle atrophy by stimulating differentiation and inhibiting myostatin expression in C2C12 myoblasts

作　　者：王会玲[1] 李燕[1] 徐凯[1] 刘楠梅[1] 田军[1] 张金元[1] 

机构地区：[1]上海解放军第四五五医院肾脏科,南京军区肾脏病研究所,上海200052

出　　处：《上海医学》2014年第5期403-408,I0005,共7页Shanghai Medical Journal

基　　金：上海浦江人才基金项目资助(10PJD016)

摘　　要：目的探讨1,25羟活性维生素D3(1,25-D3)对白细胞介素-6(IL-6)刺激下肌原细胞增殖和分化的影响及其机制。方法体外培养小鼠C2C12肌原细胞株,以2%马血清使细胞分化为肌索,给予IL-6和1,25-D3刺激细胞,观察细胞增殖情况和肌管形态。采用Western印迹法检测维生素D3受体(VDR)、生肌分化因子D(MyoD)、肌细胞生成素(myogenin)和肌动蛋白重链(MHC)的蛋白表达量;采用荧光定量聚合酶链反应(PCR)检测肌肉生长抑制素(myostatin)和肌萎缩Fbox-1蛋白(Atrogin-1)、MyoD和肌细胞生成素mRNA的转录水平。结果倒置相差显微镜下见C2C12细胞呈梭形贴壁生长,增殖迅速;以2%马血清分化培养72h后,细胞可形成条索状肌管。以20ng/mL IL-6刺激的细胞在培养48h内增殖较迅速,此后增殖缓慢;分化培养后的细胞融合形成的肌索较纤细且稀疏,直径明显缩短;以100ng/mL IL-6刺激的细胞在培养72h后有较多细胞发生脱落坏死,分化培养后的细胞形成的肌纤维极少且纤细。与对照组比较,IL-6+1,25-D3组的VDR和1,25-D3组的VDR、MyoD、肌细胞生成素、MHC的相对表达量均显著升高(P值分别<0.01、0.05),IL-6+1,25-D3组MHC和IL-6组MyoD、肌细胞生成素、MHC的相对表达量均显著降低(P值分别<0.05、0.01)。与1,25-D3组比较,IL-6+1,25-D3组MyoD、肌细胞生成素、MHC和IL-6组VDR、MyoD、肌细胞生成素、MHC的相对表达量均显著降低(P值分别<0.01、0.05)。与IL-6组比较,IL-6+1,25-D3组VDR、MyoD、肌细胞生成素、MHC的相对表达量均显著升高(P值分别<0.01、0.05)。与对照组比较,1,25-D3组MyoD mRNA表达显著升高(P<0.05),肌肉生长抑制素mRNA表达显著降低(P<0.05);IL-6组MyoD和肌细胞生成素mRNA表达均显著降低(P值均<0.01),肌肉生长抑制素和Fbox-1蛋白mRNA表达均显著升高(P值分别<0.01、0.05);IL-6+1,25-D3组肌细胞生成素mRNA表达显著降低(P<0.05),肌肉生长抑制素mRNA表达显著升高(P<0.01)。与1,25-D3组比较,IL-6+1,Objective To investigate the effect and mechanism of 1,25-dihydroxy-vitamin D3 （1,25-D3） on proliferation and differentiation of C2012 myoblasts treated with interleukin-6 （IL-6）. Methods Mouse 02012 myoblasts were cultured in vitro and induced to differentiate into myotubes by 2 % horse serum. After treated with IL-6 or 1,25-D3, cell proliferation and myotube appearance were observed by inverted phase contrast microscope. The protein levels 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 molecules （myostatin and Atrogin-1） and the mRNA levels of MyoD and myogenin were detected by fluorescent quantitative polymerase chain reaction （POR）. Results The C2C12 myoblasts grew rapidly and had a fusiform. After cultured with 2% horse serum for 72 h, the cells were differentiated into myotubes. The cells grew rapidly when treated with 20 ng/mL IL-6, while cell proliferation was slow, the size of myotubes became slim and the diameter was decreased markedly at 48 h after IL-6 exposure. Many necrotic cells were observed when treated with IL-6 （100 ng/mL） for 72 h and muscle fibers became slim. Compared with control group, the relative expression levels of VDR in IL-6 ＋ 1,25-D3 group and VDR, MyoD, myogenin and MHO in 1,25-D3 group were significantly increased （P〈0.01 or P〈0.05）, while the expression of MHO in IL-6 ＋ 1,25-D3 group and MyoD, myogenin and MHC in IL-6 group were significantly decreased （P〈O. 05 or P〈O. 01）. Compared with 1,25-D3 group, the expression of MyoD, myogenin and MHO in IL-6 ＋ 1,25-D3 group and VDR, MyoD, myogenin and MHC in IL-6 group were significantly decreased （P〈0.01 or P〈0.05）. Compared with IL-6 group, the expression of VDR, MyoD, myogenin and MHO in IL-6 ＋ 1,25-D3 group were significantly increased （ P〈O. 01 or P〈O. 05）. Compared with control group, MyoD mRNA level in 1,25-D3 group

关 键 词：蛋白质能量消耗 慢性肾脏病 C2C12成肌细胞 维生素D 白细胞介素-6 

分 类 号：R692[医药卫生—泌尿科学]