FHL1基因表达与先天性马蹄内翻足:15例患者及3名正常人的对比分析  

FHL1 gene expression and idiopathic congenital clubfoot: Comparative analysis of 15 patients and 3 normal controls

作　　者：王莉莉[1] 辛娜[2] 孙开来[2] 

机构地区：[1]中国医科大学附属盛京医院小儿外科先天畸形重点实验室,辽宁省沈阳市110004 [2]中国医科大学基础医学院遗传教研室,辽宁省沈阳市110001

出　　处：《中国组织工程研究与临床康复》2010年第7期1315-1318,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家重点基础研究发展规划(2001CB510301)项目子课题资助~~

摘　　要：背景:前期研究表明,在马蹄内翻足患者肌肉组织中存在FHL1基因表达下调,并利用凝胶阻滞实验在体外初步验证了HOXD13和FHL1基因启动子区转录因子预测位点的结合作用,但凝胶阻滞实验结果不一定能真实地反映体内转录调控蛋白和DNA结合的状况。目的:应用Western-blot技术进一步验证先天性马蹄内翻足患者足部肌肉组织中FHL1,HOXD13基因蛋白质水平的表达,应用染色质免疫沉淀技术验证在胚胎足部发育时在体内HOXD13和预测结合位点的结合作用。方法:15份先天性马蹄内翻足患儿肌肉组织标本由中国医科大学附属第二临床医院小儿外科提供,3份同年龄组正常儿童足部肌肉组织由中国医科大学法医学院提供,1例孕13周流产胚胎由中国医科大学附属第二临床医院妇产科提供。所有标本使用均经患者及其家属知情并同意。应用Western-blot方法检测15例先天性马蹄内翻足患儿及3例同年龄组正常儿童足部肌肉组织HOXD13和FHL1表达情况;并用软件预测FHL1基因上游HOXD13的结合位点,染色质免疫沉淀实验验证胚胎发育时HOXD13和FHL1的相互作用,以不表达HOXD13蛋白的脑组织作为对照。结果与结论:与同期正常儿童足部肌肉组织相比,15例人先天性马蹄内翻足患儿肌肉组织中有7例存在FHL1基因蛋白质水平表达下调,而这7例患儿中有5例同时存在HOXD13基因蛋白质表达下调。沉淀的人胚胎足部组织染色质中有预测HOXD13结合位点的扩增,无对照位点的扩增,对照脑组织中无预测位点的扩增。结果进一步验证显示了先天性马蹄内翻足患儿足部肌肉组织中FHL1、HOXD13基因表达下调;人类胚胎肢体发育过程中HOXD13蛋白可以和FHL1启动子区的预测位点结合发挥其转录调节作用。提示在人胚胎足部发育时,HOXD13表达下调导致了FHL1表达水平的下调,进而影响了足部肌肉生长发育和分化导致马蹄内翻足畸形的发�BACKGROUND： Preliminary study shows that the FHL1 gene expression is down-regulated in muscle tissue of patients with clubfoot, and the gel retardation experiments have verified HOXD13 and FHL1 gene promoter region transcription factor predicted binding sites in vitro, but the experimental results are not enough to truly reflect in vivo transcriptional regulatory protein and DNA binding conditions. OBJECTIVE： Western-blot technique is utilized to further validate FHL1 and HOXD13 gene expression in protein levels in muscle tissue of patients with congenital clubfoot, the in vivo HOXD13 and predicted binding sites in embryonic foot development was verified using chromatin immunoprecipitation technology. METHODS： Muscle tissues were samples from 15 children with congenital clubfoot, in the Department of Pediatric Surgery, at the Second Affiliated Clinical Hospital of China Medical University, 3 copies of normal children foot muscle tissues at the same age were provided by the Forensic Medicine College of China Medical University, 1 case of aborted embryo at pregnancy 13 weeks were offered by Department of Obstetrics and Gynecology, at the Second Affiliated Clinical Hospital of China Medical University. All specimens used are given informed consents by the patients and their families. Western-blot method was applied to detect the expressions of HOXD13 and FHL1 in foot muscle tissue of 15 patients of congenital clubfoot and 3 normal children at the same age; The FHL1 gene upper stream HOXD13 binding sites was predicted using software, the interaction of HOXD13 and FHL1 during embryonic development was verified with chromatin immunoprecipitation experiment, brain tissue without HOXD13 protein expression served as controls. RESULTS AND CONCLUSION： Compared with normal children foot muscle tissues, both HOXD13 gene （5/15） and FHL1 （7/15） were down-regulated in 15 patients of congenital clubfoot. An enrichment of the predicted HOXD13 binding site was observed in the precipitated human embryo foot tissues c

关 键 词：先天性马蹄内翻足 FHL1 转录调控 HOXD13 组织工程 

分 类 号：R318[医药卫生—生物医学工程]