异丙肾上腺素防治失神经骨骼肌萎缩机制的研究.docVIP

异丙肾上腺素防治失神经骨骼肌萎缩机制的研究 　　摘要目的:研究异丙肾上腺素对失神经骨骼肌萎缩中NF-κB、MuRF1表达变化的影响,探讨其防治失神经骨骼肌萎缩的机制。方法:选用wista大鼠54只,制作成标准右侧坐骨神经离断、腓肠肌失神经支配模型,随机分为对照组和药物组,应用RT-PCR和Western Bloting检测不同时段的NF-κB、MuRF1 mRNA和蛋白质表达水平,联合肌湿重分析其相关性。结果:失神经支配后腓肠肌NF-κB、MuRF1 mRNA和蛋白质表达持续增加(P0.05)。药物组肌湿重和肌细胞横截面积比对照组同时间点明显增加(P0.05),NF-κB、MuRF1 mRNA和蛋白质表达则明显降低(P0.05)。结论:异丙肾上腺素可以通过抑制NF-κB/MuRF1途径有效防治失神经骨骼肌萎缩。 　　关键词失神经骨骼肌萎缩核因子NF-κB泛素-蛋白酶体途径 肌肉环指因子-1? 　　doi:10.3969/j.issn.1007-614x.2010.10.006?? 　　AbstractObjective:To investigate the change of NF-κB and muscle Ring Finger-1(MuRF1) on denervation muscle atrophy intervated with isoprel.Methods:Levels of NF-κB and MuRF1 mRNA and protein in gastrocnemius muscle at different time after denervation were detected by RT-PCR and western Blotting.The ratio of muscle wet weight was analyzed for comparison.Result:The level ofmRNA and protein of NF-κB and MuRF1 increased correpondingly at different time after denervation(P0.05)but The group with isoprel decreased at the same time.Conclusion:the change of mRNA and protein presented consistency at different time,and MuRF1 is actively correlated to with increase of levels of NF-κB,indicating that NF-κB/MuRF1 pathway plays an importance role in denervation atrophy of the gastrocnemius,and isoprel is possible to delay denervation muscle atrophy by inhibite the pathway of NF-κB/MuRF1.? 　　Key wordsDenerbated skeletal muscule atrophy;Nuclear factor-κB;Ubiquitin-proteasomes pathway;Muscule ring finger-1(MuRF1)? 　　 　　资料与方法? 　　材料:①动物:选用成年雄性wista大鼠54只,体重180±10g,由山西医科大学生理实验室提供。②药物:异丙肾上腺素。③试剂:RT-PCR试剂盒,Trizol试剂,DNA和蛋白marker,蛋白提取试剂,NF-κB P 65(F-6)单抗、MuRF 1(H-145)多抗、β-Actin(C 4单抗)和ECL化学发光试剂,辣根酶标记山羊抗兔二抗。? 　　方法:①动物分组与失神经肌萎缩模型建立:54只雄性wista大鼠随机分为3组,即失神经对照组(A组,24只),药物组(B组,24只)和空白对照组(C组,6只),A组合B组麻醉后暴露大鼠双侧坐骨神经,左侧行假手术,右侧行坐骨神经离断,长约1.0cm,近端和组织缝合,造成右下肢失神经骨骼肌萎缩模型。空白组不做任何处理,手术当天麻醉下取双侧腓肠肌,记0天组,余每组术后2、7、14、28天随机取6只大鼠,麻醉下分离双侧腓肠肌,分别称肌湿重,提取RNA和蛋白质。②药物干预:采用腹腔注射异丙肾上腺素,剂量为5mg/Kg,每日1次。③肌湿重测定:将双侧腓肠肌完整取下,用电子天平准确称取肌湿重,计算右侧(失神经侧)和左侧(假手术侧)肌湿重比。④引物设计:基因库查询后由上海生物工程公