ABSTRACT
BACKGROUND:The oxygen free radicals and apoptosis play an important role in limb ischemia/reperfusion injury, so we can al eviate limb ischemia/reperfusion injury by inhibiting the production of oxygen free radicals and apoptosis. OBJECTIVE:To discuss the application and effect of edaravone on limb ischemia/reperfusion injury in rats. METHODS:Of the 30 female Sprague-Dawley rats, 20 rats were randomly selected to make models of limb ischemia/reperfusion injury by ligating the root of right lower limb with a self-made bal oon cuff at 40 kPa pressure to block blood flow for 4 hours and reperfusing. After success model establishment, they were randomly assigned to two groups. In the edaravone perfusion group, edaravone 3 mg/kg was injected via the left femoral vein at 5 minutes before reperfusion. In the model group and normal group (the remaining 10 rats), an equal volume of physiological saline was given at the same time point. At 24 hours after reperfusion, the right anterior tibial muscle of each group was removed and these ultrastructural changes were observed by transmission electron microscope. Bcl-2 mRNA and Bax mRNA of rat anterior tibial muscle of each group were semiquantitatively detected with the RT-PCR and the ratio of bcl-2/bax was calculated. RESULTS AND CONCLUSION:(1)Electron microscope results:compared with the model group, the muscle fibers were neater;the M line and the N line were clearer;the swel ing of mitochondria was al eviated;the numbers of mitochondria and mitochondrial crista were also increased in the edaravone perfusion group. (2)RT-PCR results:At 24 hours after reperfusion, the relative expression of bcl-2 mRNA and the ratio of bcl-2 mRNA to bax mRNA in right anterior tibial muscle were lower in the model group compared with the edaravone perfusion group (P<0.05). However, relative expression of bax mRNA was greater in the model group than that in the edaravone perfusion group, which were both higher than the normal group (P<0.05). Results indicated that the free radical scavenger edaravone relieved limb ischemia/reperfusion injury by improving the mitochondrial ultrastructure and promoting expression of bcl-2 mRNA and inhibiting expression of bax mRNA, and could provide a new choice for the treatment of limb ischemia/reperfusion injury.
ABSTRACT
BACKGROUND:Now it has cooperation and facilitative effete between myogenic regulatory factors through a long time study. So, gene therapy of double genes of Myod1 and Myog can obtain better effect, and can provide a new way for preventing denervated skeletal muscle atrophy. OBJECTIVE:To construct eukaryotic co-expression vector carrying Myod1 and Myog genes. METHODS:Ful-length Myod1 gene and Myog gene cDNA were amplified by reverse transcription PCR, and then inserted into pVAX1 vector after digested to establish the recombined Myod1 and Myog eukaryotic co-expression vector pVAX1-Myod1-IRES2-Myog-IRES2-EGFP, and then identified with gene sequencing. The in vitro cultured 3T3 cel s were transfected with pVAX1-Myod1-IRES2-Myog-IRES2-EGFP, and the expressions of Myod1 and Myog genes in the 3T3 cel s were detected with western blot assay in order to identify whether the 3T3 cel s could express the target protein correctly. RESULTS AND CONCLUSION:The sequencing results showed that the sequence length and base sequence of Myod1 and Myog cDNA in eukaryotic co-expression vector pVAX1-Myod1-IRES2-Myog-IRES2-EGFP were identical with the reported sequences. Myod1 and Myog protein band expressions were detected in 3T3 cel s by western blot after transient transfection. The pVAX1-Myod1-IRES2-Myog-IRES2-EGFP, a eukaryotic co-expression vector of Myod1 gene and Myog gene is successful y constructed.
ABSTRACT
BACKGROUND:Recent studies found that some factors play important role in the process of denervated muscle atrophy, especial y the feak-headbox transcription factor, is the key element to regulate the denervated muscle atrophy. OBJECTIVE:To investigate the effect of RNA interference on inhibiting feak-headbox 3a gene expression in vitro. METHODS:The myoblast cel line L6 were cultured in the 6-wel cel culture plates, then pEGFP-N1 and smal interfering RNA recombinant plasmid with the same ratio was transfected under the Lipofectamine2000 mediation to optimize the transfection efficiency of the detection system;2μg smal interfering RNA recombinant plasmid of feak-headbox 3a gene were transfected with myoblast cel line L6 for 48 and 72 hours. RESULTS AND CONCLUSION:At 48 hours after pEGFP-N1 and siRNA recombinant plasmid transfection, a large number of bright green fluorescent displayed under fluorescence microscope with higher transfection efficiency. Real-time quantitative PCR analysis showed that there were significant differences in the sequences of feak-headbox 3a-Ⅰ, feak-headbox 3a-Ⅱ, feak-headbox 3a-Ⅲ, feak-headbox 3a-Ⅳ on feak-headbox 3a mRNA when compared with the control group at 48 and 72 hours after trasfection (Phours after transfection when compared with that at 48 hours after transfection. Western Blot gray analysis showed that there were significant differences in sequences of feak-headbox 3a-Ⅰ, feak-headbox 3a-Ⅱ, feak-headbox 3a-Ⅲ, feak-headbox 3a-Ⅳ on feak-headbox 3a mRNA when compared with the control group at 48 and 72 hours after trasfection (Psignificantly inhibit the fork-head transcription factor feak-headbox 3a gene expression, and the inhibition effect of feak-headbox 3a gene smal interfering RNA recombinant plasmid transfected with the sequence on the mRNA and protein level of feak-headbox 3a is not clear, which can provide new idea for the gene therapy of RNA mediated denervated skeletal muscle atrophy.
ABSTRACT
BACKGROUND: Myogenic regulatory factors Myf-5 is an importance gene involved in muscle occurrence process to adjust and control, start and maintain the skeleton muscle cell growth. It has a possible relation with denervated skeletal muscle atrophy. OBJECTIVE: To detect the expression of Myf-5 gene in different skeletal muscle sitas and different periods following denervatad skeletal muscle atrophy.DESIGN, TIME AND SE'I-rlNG: Randomized control animal experiment was performed in the Shanxi Medical University between March and April in 2008. MATERIALS: A total of 24 Sprague-Dudley male rats was selected and divided randomly into four groups, with six in each, Le. sham operated group (innervations), denervated 2 d group, denarvated 7 d group and denervatad 28 d group. METHODS: Sham operations were made only for the rats in the sham operated group and sciatic nerve were not cut off. Sciatic nerves were cut off more than one centimeter at the mid-level of their right lower limb for the rats in denervated groups. The rats were executed by vertebrae dislocation method at 2, 7, 28 days after denervation. The skeletal muscles of the right lower limb (tibialis anterior, soleus, gastrocnemius and plantar) were dissected and dissociated. MAIN OUTCOME MEASURES: Myf-5 mRNA expression was detected by reverse transcription-polymerase chain reaction. Immunohistochemical stain (ABC method) with polyclonal antibody against Myf-5 protein was performed and the gray value was counted.RESULTS: Expressions of Myf-5 mRNA in denervatad skeletal muscle were up-regulated at 2, 7, 28 days in the eady stage of denervation (P < 0.05). The number of Myf-5 antibody positive-stained cell nuclei was the most in satellite cells at 28 days following denervation. CONCLUSION: At the early stage of denervated skeletal muscle atrophy in SD rats, expressions of Myf-5 in different skeletal muscles are all up-regulated. Denervation of rat skeletal muscle raises the Myf-5 expression in satellites cells.
ABSTRACT
BACKGROUND: MyoD expression of skeletal muscle of rats increases distinctly in the earlier period of denervation, which can greatly postpone denervated skeletal muscle atrophy. The clinical test testifies that electrical stimulation is an effective method to cure denervated muscle atrophy. But the influence of electrical stimulation on MyoD expression during denervated muscle atrophy is still unproved. OBJECTIVE: To discuss the influences of electrical stimulation on the gene expression of MyoD of skeletal muscle of rats. DESIGN, TIME AND SETTING: Randomized control animal experiment was performed in the Animal Experimental Center of Shanxi Medical University between July and November in 2008. MATERIALS: A total of 36 healthy Sprague-Duwley rats of either gender were divided randomly into three groups, control group, denervation group and electrical stimulation group. Each group contained 12 rats. METHODS: Standard models of right sciatic nerve dissociation and gastrocnemius denervation were established in right limb of each rat in denervation group and electrical stimulation group. Thirty-minute electrical stimulation was given to the denervated gastrocnemius muscle of each rat of electrical stimulation group once a day. The rats were executed at 2, 7, 14, 28 days after denervation to dissect and dissociate their gastrocnemius muscles of the right lower limbs. MAIN OUTCOME MEASURES: MyoD mRNA expression was detected by reverse transcription polymerase chain reaction, and MyoD protein level by immunohistochemistry. RESULTS: The expression of MyoD mRNA and MyoD protein level in specimens of denervation group and electrical stimulation group were up-regulated at 2, 7, 14, 28 days after denervation, with significant differences compared with blank control group (P