| Objective : Duchenne muscular dystrophy(DMD),the most common form of muscular dystrophy,is a lethal and incurable genetic muscle muscle wasting disease.DMD is caused mainly by mutations in the DMD gene on the X chromosome(Xp21.2),which encodes the dystrophin protein.Dystrophin is located on the intracellular surface of the sarcolemma,and it constitutes the structural cornerstone of muscular cells.The absence of dystrophin protein,leading to recurrent muscle fiber damage during contraction.The main clinical manifestation of DMD is progressive muscle weakness,atrophy and sham of the gastrocnemius muscle hypertrophy.Eventually,the loss of dystrophin results in inability to walk about 12 years old.Ultimately,die of respiratory and cardiac failure,and death before the age of 20 years.The main pathological features of DMD patients is the unequal size of muscle fiber diameter,progressive necrosis,inflammation,and fibrosis.Due to absence of dystrophin protein,causes the dystrophic phenotype,Chronic inflammation plays a key role in the progressive muscle degeneration observed in the pathogenesis of DMD.The progressive muscle injury is exacerbated by the inflammatory response of a heterogeneous infiltrate of immune cells.However,in the early stage of DMD,the abnormal activation of inflammatory signaling pathway has occurred In the early stage of DMD,before the onset of muscle fiber necrosis.In recent years,increasing attentions have shown that inflammatory response and activation of inflammatory pathways play an important role in the occurrence and progression of DMD.The regulation of inflammatory response is an important therapeutic method for DMD therapy.The dystrophin-deficient C57BL/10ScSn-Dmdmdx/J(mdx)mice are the most widely application animal model for DMD.Due to the mutation of exon23 in X chromosome,which causes the dystrophin absence reflects in as modulation of skeletal muscle.Many studies have been done to develop treatments for DMD,such as gene therapy.To correct the gene defects or play a role of treatment Gene therapy can delivery the normal gene or therapeutic genes into target cells.Adeno-associated virus AAV is one of the most commonly used gene therapy vectors for gene therapy.AAV can expressed efficiently for a long time,and has the advantages of no toxicity,no cell immunity and no pathogenicity.Systematic evaluation showed that AAV9 could be effectively transfected into skeletal muscle and myocardium.Insulin-like growth factor 1 IGF-I is a potent signal for growth in a number of tissues.IGF-I is a nutritional factor which is important in the process of cell proliferation,differentiation and maturation.In addi-tion,the enhanced IGF-1 expression in skeletal muscles significantly diminished the amount of fibrosis,decreased myonecrosis,increased the rate ofmuscle regeneration,reduce serum CK and improve muscle strength.Protein kinase B(PKB or Akt)activation has been shown to be a major component of how IGF-I mediates cell survival and growth in muscles.Interestingly,IGF-1 repressed the expression and activity of MIF,HMGB1,and NF-?B.Previous studies have focused on the effects of IGF-1on muscle fiber regeneration,and little attention has been paid to the role of IGF-1 in muscle inflammation.Considering that DMD is a kind of myopathy involved almost every muscle in the body,the tail vein injection is more suitable for clinical application.Our study using the vector AAV9 carrying hIGF-1 gene into the 6 week old mdx mice by a single injection and the AAV9 carrying GFP as control.The inflammatory response was evaluated by routine histopathological staining,immunofluorescence and Western blot 6 weeks later.Furthermore we explore the mechanism of hIGF1 effect on inflammatory response.Methods: Given the experimental group mdx mice tail vein injection ofAAV9-hIGF-1 virus 200 L(1 * 1012vg/ml),experimental control group mdx mice tail vein injection of AAV9-GFP virus 200 L(1 * 1012vg/ml),after 6weeks were observed in mice receiving virus after intravenous injection under the same conditions.Clipped the skeletal muscle and myocardium after mice were anesthetized respectively.Under fluorescence microscope we observed the expression of AAV9 in muscle.Next step,We observed the expression of hIGF-1 protein.HE staining and ACP staining were used to observe the changes of inflammatory response.The expression of inflammation related proteins CD68,PP65 were Detectd.Data were analysed by spss 13.0.Results:1 The expression of AAV9 in mdx mice.GFP were expressed in different tissues of mdx mice.The tibialis anterior muscle expressed the most,and then the triceps muscle,other organizations only expressed a small amount.2 The expression of AAV9-hIGF-1 in the tibialis anterior of mdx mice.In the tibialis anterior of mdx mice,high-level expression of AAV9-hIGF-1 were obtained,but there is still no expression of muscle fibers.3 The inflammatory response of mdx mice after AAV9-hIGF-1therapyAAV9-GFP group was observed a a mixture of inflammatory cells infiltration,while in AAV-9hIGF-1 group was observedi a small amount of inflammatory cells infiltration by HE staining and ACP staining.CD68 is a reliable marker of macrophage.AAV9-hIGF-1 group of CD68+cells was lower than AAV-GFP groupStatistical analysis of percentage of the inflammatory area,AAV-hIGF-1group(1.78 + 0.47%),lower than that of group AAV-GFP(3.4 + 1.22%),higher than the normal group(0%),the differences were statistically significant(p<0.05).4 The change of NF-kappa B signaling pathway.We observed AAV9-hIGF-1 reduced the expression of PP65.AAV-hIGF-1 group(0.45 + 0.07%),lower than that of group AAV-GFP(0.76+ 0.13%),higher than the normal group(0.38 + 0.06%),by Western-blot.The difference was statistically significant.Conclusions:1 AAV9 can be expressed in multiple sites of mdx mice through the tail vein injection,the anterior tibial expressed the most.2 hIGF-1 intravenous injection therapy can relieve the inflammation of the muscles of mdx mice.3 The anti-inflammatory action of hIGF-1may throught down regulation of NF-kappa B. |
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