【作者】 张磊；

【导师】 梁炳生；

【作者基本信息】 山西医科大学 ， 外科学， 2010， 硕士

【摘要】 目的研究异丙肾上腺素、对核转录因子kappaB (nuclear factor of kappaB, NF-kappaB)p65和E3连接酶MAFbx活性的影响,探讨各种药物防治肌萎缩的作用及其机制方法选择健康的雄性SD大鼠54只,体重195±5g,随机分为A,B,C,D,E,F,G,H,I 9组,每组6只。A组为对照组(假手术组),B、C、D,E组为去神经组,F、G,H,I为异丙肾上腺素组。大鼠分批用戊巴比妥钠(40mg/kg, ip)麻醉后,用右下肢后部中段切断坐骨神经的方法制作失神经骨骼肌动物模型,正常组仅做假手术(不切断坐骨神经),去神经组及异丙肾上腺素组切断坐骨神经1cm以上,分别于去神经第2d、7d、14d、28d处死大鼠(用脊椎脱臼法),用显微外科技术认真游离失神经腓肠肌,切取的肌肉样本快速置于-80℃冰箱冻存,备用。用RT-PCR法检测MAFbx与NF-KB的mRNA:以GAPDH作为内参照,做半定量检测。取样本组织50mg匀浆后用上海生工抽提试剂进行总RNA抽提,然后用MBI的RT-PCR试剂盒,按样本RNA2μg,20μl体系进行反转录。MAFbx其上游引物：5’cta cga tgt tgc agc caa ga 3’,下游引物：5’Ggc agt cga gaa gtc cag tc:3’,共167bp；P65的上游引物：5’gac cag gaa gtc agc gag tc3’下游引物：5’tcc aga ggg aca get ctt gt3’共176bp.作为内参照的GAPDH的引物由山西医科大学实验中心提供,其上游引物：5’tga acg gga agc tca ctg g 3’,下游引物：5’tcc acc acc ctg ttg ctg ta 3’,共307bp.聚合酶链反应按50μl体系,反应条件为：96℃,2′, 1cycle。96℃,30″; Tm(Myf-5 53℃)/(GAPDH 50℃),30″; 72℃,2′,36 cycle。用免疫印迹法(Westernblot法)测定MAFbx与NF-KB的蛋白质：用β-actin作为内参照,做半定量检测。取样本组织100mg匀浆后用普利莱蛋白提取试剂盒进行总蛋白抽提,煮沸、离心后,加样于垂直电泳槽行SDS-PAGE,电泳约3h,转膜1小时30分钟,封闭室温4小时,孵育一抗(Ab1)4℃过夜,漂洗后孵育二抗(Ab2)4℃约4小时,TBS漂洗后,置于暗室加ECL发光试剂盒反应,一分钟左右立即放在暗箱曝光,60秒左右取出浸入显影剂内,观察至最佳显影后,捞出在清水中洗净,然后置于定影剂中15分钟,取出晾干后用数码相机获取图片。最后用NIH的ImageJ软件获取数据,用SPSS软件进行统计分析。结果去神经骨骼肌萎缩时,腓肠肌MAFbx、NF-KB的mRNA和蛋白质在去神经支配后第2d、7d、14d、28d不同时段表达均为上调。NF-KB药物各组与失神经组同时段比较均下调(除2d组外)P<0.05,而MAFbx表达无统计学意义,P>0.05.结论大鼠失神经骨骼肌萎缩早期,MAFbx、NF-KB的表达在腓肠肌为上调,异丙肾上腺素是通过NF-KB途径来防治失神经早期的骨骼肌萎缩的。更多还原

【Abstract】 Objective To study the influence of Isoprenaline on the expression pattern of nuclear factor of kappaB (NF-kappaB) and muscle atrophy F-box MAFbx and explore the preventive and therapeutic effect and mechanisms of Isoprenaline on denervated skeletal muscle atrophy.Method A total of 54 healthy male and 8w age Sprague-Duwley rats were selected. They have 195±5g weight and were devided randomly into nine group, A control group (innervations)、experimental B group (denervated 2d)、experimental C group (denervated 7d)、experimental D group (denervated 14d), experimental E group (denervated 28d) Isoprenaline F group (denervated 2d)、Isoprenaline G group (denervated 7d)、Isoprenaline H group (denervated 14d), Isoprenaline J group (denervated 28d) six rats in every group. After the rats were anesthetized by pentobarbital sodium (40mg/kg) intraperitoneal injection in batch, The midpiece_sciatic nerve of their right lower limb posterior part were cut off for making animal model of denervated skeletal muscle. Sham operation was made only for the rats in normal group (sciatic nerve were not cut off) and sciatic nerve were cut off more than one centimeter for the rats in denervated group. The rats were executed by vertebrae dislocation method in batch. The rats in control group and experimental a group were executed in 48 hour after operation, experimental B and F group for seven day, experimental C and G group for twenty-eight day and so on. whereafter muscle specimen were quickly placed in -80℃refrigerator for reserve. MAFbx and NF-kappaB mRNA level detected by RT-PCR: GAPDH,as a inner reference, are used to make semiquantitative detection for Myf-5mRNA.Take 50mg sample tissue to homogenate, and then extract total RNA by extract reagent from shanhai sangon,next to have it inversely transcripted by RT-PCR reagent box from MBI according as 2μl sample RNA and 20μl system. MAFbx primer derives from primer5.0 software design. the upper stream primer is 5’cta cga tgt tgc agc caa ga 3’, the downstream primer is 5’Ggc agt cga gaa gtc cag tc3’, 167bp.P65:5’gac cag gaa gtc agc gag tc 3’. the downstream primer is:5’tcc aga ggg aca get ctt gt 3’,176bp.As a inner reference,GAPDH primer come from Shanxi medical university molecule experiment centre and its piece length have 308bp. polymerase chain reaction goes on by 50μl system and its reaction condition as follow:96℃,2’,1cycle。96℃-30″; Tm(Myf-5 53℃)/(GAPDH 50℃)-30″; 72℃,2′,36 cycle. MyoD protein level detected by Westernblot:β-actin,as a inner reference, are used to make semiquantitative detection for MAFbx and NF-kappaB protein. Take 100mg sample tissue to homogenate, and then extract total protein by protein extract reagent box from Applygen Technologies Inc. after boiled for 5min and centrifugated in sample buffer for 10 min,RNA samples were spotted into pore of vertical electrophoresis bath and were separated in 12% SDS-PAGE for about three hour and transferred onto nitrocellulose membrane (Bio-Rad) for 1 hour and thirty minutes.Nonspecific reactivity was blocked by incubation 4h at 20℃in buffer(10mM Tris-HCl, pH7.5,150mM NaCl, 2%Tween-20,4% bovine sreum albumin).After floating in TBST buffer, The membrane was then incubated with primary antibody overnight at 4℃. After floating in TBST buffer, The secondary antibody was used to incubated with the membrane for 4h at 4℃. After floating in TBST buffer and TBS buffer, The membrane was then placed in dark room and reactive protein was detected by ECL chemiluminescence system(santa cruz). the membrane was immediately placed into camera obscure for exposition about 1 min,and was taken out to immerse into developer until developing was optimization. then the membrane was immediately got out of the developer, placed into fixerfor about fifteen minutes, taken out for open-air drying, photographed by figure camera for store.Finally,ImageJ software from NIH was used to get image data about mean grey value.then SPSS software-was used to statistics ordered data by one-way anova analysis. Sigmaplot software was used to statistics plotting.Result Expression of MAFbx、NF-κB mRNA and protein all are up-regulation at 2d、7d、14d、28d after denervation. The effect shows that only the expression of NF-κB mRNA and protein level in specimens of Isoprenaline group are down-regulated at 2d、7d、14d、28d after denervation compared with that in denervation Group (P<0.05).Conclusion:Expression of MAFbx、NF-κB were up-regulation at 2、7、14、28 days after denervation In the early stage of skeletal muscles.only NF-κB expression of gastrocnemius denervation model of rats can be down-regulated by Isoprenaline, which shows that Isoprenaline is a effective way of delaying denervated skeletal muscle atrophy through participate in NF-κB path.更多还原