The Inflammation And The Expression Of Mpeg 1 In Mdx Mice Skeletal Muscle

Objective:Progressive muscular dystrophy(PMD) is a group of progressive increase in muscle weakness,muscle atrophy of skeletal muscle disorders collectively hereditary main clinical manifestations. Since the gene encoding muscle cells with mutation related proteins,resulting in protein or dysfunction,causing damage to the integrity of the muscle cells. The morbidity of Duchenne muscular dystrophy(DMD) in various types of muscular dystrophy is the highest,which is 1/3500 baby boy and no significant differences between regions and races.DMD is an X-linked recessive genetic disease,the causative gene locates on the X chromosome Xp21, and endodes dystrophin protein.It occurs deletion、insertion and point mutations, dystrophin protein loss of function,and results muscle fiber degeneration and necrosis. Mostly female carriers of disease genes,the incidence is rare and mild. Because the age of DMD is small, insidious onset and high mortality, it has been widely concerned.Currently,the pathogenesis of DMD is unclear, which involves a lot of common mechanisms,For example,calcium influx,inflammatory cells infiltration, activation of proinflammatory cytokines and promote fibrosis, activation of various proteolytic enzymes, the defection of autophagy and apoptosis, etc. Studies have shown that local inflammation is the trigger point of muscle fibrosis and adipose tissue penetration,resulting in muscle cells induced by adipose tissue replacement, loss of function. In the inflammatory response,macrophage infiltration plays a key role,such as activated macrophages can produce NO cracking muscle cell, at the same time as the main proinflammatory factor, inhibit muscle regeneration by activating protein hydrolysis system and cause the muscle atrophy.Macrophage expressed gene 1(mpeg1) was first identified as a gene with expression tightly restricted to human and murine macrophages. Previous experiments have shown that in zebrafish into mpeg1 gene significantly increased after macrophage cell lin response,which makes neutrophile and macrophages to be studied separately,indicating mpeg1 specifically expressed in macrophages.C57BL/10 Sc Sn Dmd mdx/JNju mouse(X-linked musclar dystrophy mouse,X-chain muscle atrophy in mice,called mdx mice)for encoding the protein dystrophin gene spontaneous mutation 3185, so that the locus encoding glutamine password becames a stop codon, resulting in premature termation of the expression of the gene,which encodes a protein causing shortened.The model mice has the same genetic basis with DMD patients,and has been widely used in various fields of DMD research.For this study, we hope to observe the pathological changes in skeletal muscle of mdx mice by different staining method,use dynamic expression of the gene chip technology to screen inflammation-related genes in different stages of the disease, futher more observe macrophage expressed gene 1 by real-time quantitative PCR(q RT-PCR) technique in mdx mice and control mice.To investigata the role of macrophage-related inflammation in mdx mice,and then through the inhibition of macrophage-related inflammation to improve muscle mass, provide new therapeutic targels for DMD.Methods:Male C57BL/10 Sc Sn Dmdmdx/JNju mices were used as the experimental group,male C57BL/6Sc Sn mice were used as the control group,all mices were divided according to age 2 week、4week、8week and 12 week.Each group selected six different time points,three for routine staining,three for gene microarray and q RT-PCR determination.After the intraperitioneal injection of 10% hydration aldehydes,taken mice quadriceps, gastrocnemius, biceps and the tibialis anterior muscle, myocardial, embedding in the processing. Set the slice thickness is 8 um, serial section. On frozen sections were stained with routine,including HE staining,MGT dyeing,ACP staining morphological changes, observed its morphological chages under microscope,summed mdx mouse skeletal muscle inflammatory pathology. After the mices were anesthetized,taking into mouse quadriceps quickly frozen in liquid nitrogen,then stored at-80 ℃refrigerator for m RNA extraction, q RT-PCR detection of mpeg1 and gene chip testing,the result of applying the data spss 13.0 analysis.Results:1 Using conventional staining,we observed 2w of mdx mice quadriceps, gastrocnemius, tibialis anterior muscle and biceps muscle cells are basically the same size, and occasionally highly stain of muscle fibers,on muscle cells necrosis,inflammatory cell infiltration; 4w visible macrophage phagocytosis scattered, occasional special treatment like distribution;8w inflammatory cells infiltrate when integrated into the film,12 w when the area was reduced inflammatory lesions; mdx mice myocardial degeneration was not seen necrosis and inflammatory cell infiltration; we have not see all these pathological changes in age-matched control miceskeletal muscle;2 Useing gene chip technology for measuring m RNA mdx mice quadriceps were screened genes related to inflammation of more than 30,the results showed that compared with 2w,progress increases with the expression of genes related to inflammatory disease,while at 8w paeked,12 w decline,but still elevated when compared with 2w,the difference was statistically significant(P<0.05);3 q RT-PCR technique utilizing mpeg1 gene quantitative analysis showed that when 2w,between mdx mice and control mice mpeg1 undifferentiated content; from 4w mdx mice gradually increase the amount of mpeg1,the highest level was at 8w, with control rates among the same age difference was statisically significant(P<0.05),but the difference of the expression of mdx mice 4w,8w,12 w was not statistically signifacant;but there was no statistically significant difference between the control mice each week.Conclusions:1 Inflammation involved in the development of the disease mdx mice :beginning to emerge from 2 week,peak at 8 week,12 week has stabilized;2 Mpeg1 play a certain role in the inflammatory response in mdx mice.