Expression Of MyoD In Denervated Skeletal Muscle Of Rats

Objective To investigate expression state of MyoD in different skeletal muscle and different period at the early stage of denervated skeletal muscle atrophy.Method A total of 24 healthy male and 8w age Sprague-Duwley rats were selected.They have 195±5g weight and were devided randomly into four group,i.e control group (innervation),experimental A group(denervated 2d),experimental B group(denervated 7d),experimental C 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 were 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 24 hour after operation,experimental B group for seven day,experimental C group for twenty-eight day. Microsurgical technique was used to dissociate the leg skeletal muscle of the right lower limb (tibialis anterior,soleus,gastrocnemius,plantar),whereafter muscle specimen were quickly placed in -70℃refrigerator for reserve.MyoD mRNA level detected by RT-PCR: GAPDH,as a inner reference,are used to make semiquantitative detection for MyoD mRNA.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.MyoD primer derive from primer5.0 software design and its sequence is from 175 to 316 in MyoD mRNA full sequence.its piece length is 151bp.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:94℃,2',lcycle。94℃-30";Tm(MyoD 53℃)/(GAPDH 50℃)-30";72℃,2',36 cycle.Then take agarose gel horizontal strip electrophoresis with 160my voltage and get image through gel formatter in the end.MyoD protein level detected by Westernblot:β-actin,as a inner reference,are used to make semiquantitative detection for MyoD 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 denerved skeletal muscle(tibialis anterior,soleus,gastrocnemius,plantar)MyoD mRNA and protein all are up-regulation at 2d,7d,28d after denervation. Denervated group comparison with control group by group comparison method of analysis of variance(Tukey HSD)all are significant,P＜0.01,and denerved group is double higher than control group,difference is notable.At every time stage,expression of MyoD mRNA and protein in four muscle(tibialis anterior,soleus,gastrocnemius,plantar)make a group comparision by turn with Tukey HSD method and only some have difference,P＜0.05, difference among group is less than 10%。Conclusion On the early stage of denerved skeletal muscle atrophy in SD rat,expression of MyoD in fast muscle(tibialis anterior,gastrocnemius,plantar)and slow muscle(soleus)all are up-regulation significantly;At different time stage,expressions of MyoD in defferent skeletal muscle have partly statistic defference.In the couse of skeletal muscle,MyoD may involve in genic regulation in the molecule cell pathway and delay the process of skeletal muscle atrophy.The mechanism need to be lucubrated by step.