Smad4基因siRNA干扰对小鼠骨骼肌急性钝挫伤愈合的影响  

The Effect of Smad4 Small Interfering RNAs in Injured Mouse Skeletal Muscle after Acute Contusion

作　　者：李宏云[1] 陈疾忤[1] 陈世益[1] 陈始秋[1] 张庆国[1] 

机构地区：[1]复旦大学运动医学中心复旦大学附属华山医院运动医学与关节镜外科,上海200040

出　　处：《中国运动医学杂志》2012年第7期569-576,共8页Chinese Journal of Sports Medicine

基　　金：国家自然科学基金(30800543)资助

摘　　要：目的:了解以慢病毒为载体的Smad4 siRNA转染小鼠急性钝挫伤骨骼肌的转染效率以及抑制骨骼肌纤维化、减轻疤痕生成、促进骨骼肌损伤修复的能力。方法:用自制打击装置将54只雌性C57bl小鼠(体重20～25g)右侧腓肠肌中段造成急性钝挫伤模型,随机分为①PBS对照组、②Smad4siRNA注射组、③scrambled siRNA注射组三组,每组18只。小鼠急性钝挫伤伤后10 d,分别在三组损伤部位注射PBS、Smad4 siRNA、scrambled siRNA各0.1 ml。分别于伤后第28 d处死各组小鼠取材,荧光显微镜下观察局部绿色荧光蛋白(Green Fluorescent Protein,GFP)表达,在损伤部位取材骨骼肌组织行Real Time-PCR和Western blot方法检测Smad4基因和蛋白表达,Masson染色以及Vi-mentin免疫组化检测各组小鼠急性钝挫伤后腓肠肌局部纤维化及疤痕生成情况,生理学方法检测各组小鼠骨骼肌强直收缩和快速颤搐收缩能力,了解小鼠损伤骨骼肌愈合情况。结果:①scrambledsiRNA注射组和Smad4 siRNA注射组小鼠损伤骨骼肌部位均可检测到GFP表达,Smad4 siRNA注射组肝脏组织内并未检测到GFP表达;②Smad4 siRNA注射组损伤部位骨骼肌Smad4基因和蛋白表达明显降低;③Smad4 siRNA注射组骨骼肌疤痕生成以及vimentin表达明显低于scrambled siRNA注射组和PBS对照组;④Smad4 siRNA注射组小鼠腓肠肌强直收缩和快速颤搐收缩明显高于PBS对照组和scrambled siRNA注射组。结论:以慢病毒为载体的Smad4 siRNA可成功转染小鼠急性钝挫伤骨骼肌,并长期发挥抑制Smad4基因及蛋白表达的作用,并抑制骨骼肌损伤后纤维化,减轻疤痕生成,改善骨骼肌损伤后的愈合质量,促进骨骼肌损伤修复。Objective To determine the efficiency of lentivirusmediated Smad4 siRNA transfect into the mouse skeletal muscle and its capability of suppressing fibrosis, scar formation and improving func tional recovery of the muscle. Methods A muscle acute contusion model was utilized to hit the mouse＇s right gastrocnemius muscle for our experiment. 54 female C57bl mice （2025g） were randomly assigned to 1 of 3 groups：PBS control group,Smad4 siRNA injection group and scrambled siRNA injection group （16 each）. 0.1ml of culture medium containing PBS,Smad4 siRNA or scrambled siRNA was injected in to the contunded area 10 days after injury. The mice were sacrificed 28 days after contusion,the right gastrocnemius muscle were isolated and prepared for use. The green fluorescent protein （GFP） expressionand distribution in each group were observed under the fluorescence microscope. Realtime PCR and Western blot were used to analyze Smad4 expression at the contunded area in vivo both at gene and pro tein level. Masson trichrome staining and immunohistochemical analysis of Vimentin were did to identify the fibrosis and scar formation at local injured area. Moreover,we tested the fasttwitch and tetanus strength of the gastrocnemius muscle to determine the functional recovery of injured muscle. Results （1） The GFP detected in Smad4 siRNA and scrambled siRNA injection group after injury demonstrated that the transfection of lentivirusmediated Smad4 siRNA was successful. Meanwhile,the expression of GFP in the liver of Smad4 siRNA injected mice was negative; （2）The expression of Smad4 both at gene and protein level were suppressed obviously in the injured skeletal muscle of Smad4 siRNA injection group; （3）Masson trichrome staining and vimentin immunohistochemical analysis revealed that the scar formation and fibrosis of injured skeletal muscle were inhibited obviously in the Smad4 siRNA group compared with the scrambled siRNA group and PBS control group with statistical significance; （4）The results of tetanic

关 键 词：骨骼肌 钝挫伤 修复 SIRNA 转染 

分 类 号：R87[医药卫生—运动医学]