Mdx小鼠骨骼肌纤维化及Spp1基因表达

摘要

目的：通过观察Duchenne型肌营养不良的模型鼠mdx小鼠骨骼肌中纤维化情况，研究Spp1基因在mdx小鼠及其对照鼠中不同时期的表达，探讨在mdx小鼠中Spp1与肌纤维化的关系。方法选取雄性C57BL/10ScSn-Dmdmdx/JNju鼠为实验组，雄性C57BL/6ScSn小鼠为对照组，根据年龄分为2w组、4w组、8w组、12w组。每组选取6只，3只用于冰冻切片的苏木精-伊红染色及马松（masson）染色，3只用于基因芯片及qRT-PCR。结果2w及4w时mdx小鼠无明显结缔组织增生，8w时，mdx小鼠可见轻度结缔组织增生；12w时，mdx小鼠结缔组织增生程度较8w稍加重，仍为小片状区域的纤维化,对照组小鼠不同时期均未见纤维化；mdx小鼠2w与12w股四头肌基因芯片表达谱对比，其中Spp1基因在mdx小鼠2w与12w相比fold-change值为-15.1354，变化明显； Spp1在mdx小鼠股四头肌不同时期表达量比较：8w组较4w组明显升高，12w组较8w组表达量下降，但仍高于4w组，8w组与12w组Spp1表达量较同期对照组明显升高，差异具有统计学意义。结论 mdx小鼠早期（2w~4w）肌纤维化表现不明显，8w时骨骼肌内可见少量结缔组织增生，随后缓慢进展。②Spp1基因在mdx小鼠成熟期（8w~12w）表达量明显增加，推测其在mdx小鼠肌纤维化中发挥一定作用。%Objective To observe Duchenne type muscular dystrophy model mouse mdx mice, our experiment focuses on the mdx mice skeletal muscle fibrosis and Spp1 genes expressed in mdx mice in different periods and the control mice to further explore the relationship between Spp1 and fibrosis in DMD. Methods C57BL/10ScSn-Dmdmdx/JNju mice were used as the experimental animals and C57BL/6ScSn mice served as control group. There were four groups: 2 weeks group,4 weeks group,8 weeks group,12 weeks group .Each group included six mice for conventional histochemical staining, gene chip detection and qRT - PCR. Muscle biopsies were done for staining,including hematoxylin-eosin staining,masson staining to observe skeletal muscle fibrosis and other pathologic changes. We compaired gene expression profile in quadriceps of mdx mice different periods by gene microarray analysis. Additionally, qRT - PCR were used to detect the Spp1 gene expression in mdx and control mice quadriceps at the same time.Results Fibrosis in mdx mice at different periods individual degenerative muscle fibers can be saw occasionally in the quadriceps femoris muscle of 2w mdx mice and 4w muscle specime without obvious connective tissue hyperplasia; only partial region with slightly broadening gap between muscle fibers begin to occur at 8w,endomysial connective tissue proliferation fibers in partial region at 12w;Gene expression profile in muscle of mdx mice different periods by gene microarray analysis.Comparison of gene expression profile of mdx mouse quadriceps the fold-change of Spp1 gene at 2 weeks versus 12 weeks is 15.1354 which means the change is very obvious;The expression of Spp1 gene In the skeletal muscle of mdx mice and control mice at different periods.8 weeks group is significantly higher than the 4 weeks group,the expression of Spp1 gene is decreased in 12 weeks group compared with the 8 weeks group,but still higher than 4 weeks. The expression of Spp1 gene in the skeletal muscle of mdx mice at different periods compared with control mice. The expression of Spp1 in 8weeks group and 12weeks group is significantly higher than that in the control group,The difference was statistically significant.Conclusion ①Fibrosis is not obvious in the muscle of mdx mice at early stage(2 weeks-4weeks) and then slowly progress.A small amount of connective tissue hyperplasia can be seen in skeletal muscle at the age of 8 weeks.②The expression of Spp1 gene increased significantly in mature mdx mice(8 weeks-12 weeks),inferencing that Spp1 plays a role in muscle fibrosisof mdx mice.