The Effect Of ADM In Skeletal Muscle Ischemia Reperfusion Injury

Objective:To construct adrenomedullin eukaryotic expression vector pVAX1-ADM,then injected to rat gastrocnemius,judge the protective effect by detecting malondialdehyde content and super oxide dismutase activity in gastrocnemius,creatine kinase content and lactate dehydrogenase content in serm,and take histomorphology examination of gastrocnemius.Methods:1.Extract rat adrenal total RNA then obtain ADM gene by RT-PCR method.Clone ADM gene into the T-vector,subcloned it into the eukaryotic expression vector pVAX1 further.2.32 male SD rats,weight 200±10g,according to the weight were randomly divided into 4 groups:empty plasmid group(pVAX1),300μg dose,500μg and 700μg dose recombinant plasmid(pVAX1-ADM) group.The four groups gastrocnemius were injected recombinant plasmid for four weeks,the dose of recombinant plasmid respectively was 0μg,300μg,500μg and 700μg,once a week.RT-PCR and immunohistochemical study examination,determination of the recombinant plasmid transcription levels and protein expression levels in gastrocnemius,clearly the optimal dose of recombinant plasmid.3.32 male SD rats,weight 200±10g,according to the weight were randomly divided into 4 groups:Normal control group,IR model group, Positive medicine group(Verapamil group),Recombinant plasmid group.Before operation,Recombinant plasmid group were injected 700μg recombinant plasmid to gastrocnemius and the other three groups were injected the same capacity of normal saline,once a week.After 4 weeks,set up Ischemia-reperfusion model,ischemicing last 3h,then reperfusing last 2h.In operation,verapamil solution(2mg/kg) was injected to intraperitoneal in Verapamil group,the other three groups were injected the same capacity of normal saline,10 minutes before reperfusion.After operation,check the contents of malondialdehyde and super oxide dismutase in gastrocnemius,the contents of creatine kinase and lactate dehydrogenase in serm;take histomorphology examination of gastrocnemius.Results:1.Obtained ADM gene sequence is uniform compare with GeneBank rat ADM sequence.Identify the eukaryotic expression plasmid by PCR XbaⅠ,HindⅢdigestion,results showed that the recombinant plasmid was positive.Spectrophotometry was used to judge the recombinant plasmid,results is OD260=0.901,OD280=0.487,OD260/280=1.850. Recombinant plasmid concentration is 4.5mg/ml.2.Detection of gene expression in rat gastrocnemius muscle: semi-quantitative RT-PCR and immunohistochemistry experiments results showed that in the ADM expression was increased.3.Compared with the IR model group,the content of MDA in pVAX1-ADM group in gastrocnemius decreased 32.21%(p＜0.01), activity of SOD increased by 61.69%(p＜0.05),content of CK in serm reduces 50.69%(p＜0.05),content of LDH reduced 28.38%(p＜0.05). Compared with the verapamil group,the content of MDA in pVAX1-ADM group in gastrocnemius increased 8.91%(p＞0.05); activity of SOD increased 23.71%(p＞0.05),content of CK in serm increased 20.61%(p＞0.05),content of LDH increased 36.17%,there is statistical significance(p＜0.05). Compared with the IR modle group,the constrcture of skeletal muscle in PVAX1-ADM group and the verapamil group showed that a small amounts of skeletal muscle fibers are ruptured and infiltration of inflammatory cells skeletal muscle edema and interstitial edema are reduced significantly.Conclusion:1.ADM gene was connected to the the eukaryotic expression vector successfully.Recombinant plasmid concentration is 4.5mg/ml.2.Recombinant plasmid was injected to gastrocnemius which increase the expression of ADM.The 700μg dose recombinant plasmid group is the best expression group.3.Rat skeletal muscle ADM expression was enhanced which can reduce injury of the muscle tissue in the process of ischemia reperfusion.