摘要
目的三维黏附培养下获得并分析弥漫性特发性骨质增生症 (diffuse idiopathic skeletal hyperostosis, DISH )骨化相关特异性miRNAs。方法2012年1月至2014年1月通过手术分别获取4份DISH患者骨化黄韧带组织块与4份创伤患者正常黄韧带组织块,采用胶原酶消化法分离组织块中成纤维细胞,在人完全脱细胞羊膜 (human acellular amnioticmembrane, HAAM)上培养,收获前进行细胞免疫荧光鉴定;提取细胞总RNA并检测其质量,通过YM-100(Millipore)微离心过滤柱得到片段〈300nt的小RNA,采用μParaflo^TM miRNA微阵列基因表达实验和分析技术分析miRNAs表达谱,对结果中部分差异miRNAs进行qRT—PCR验证;采用PicTar2005、mirandav5、TargetScan5.1软件预测靶基因;使用Gene Ontol—ogy进行靶基因功能注释,基于KEGGPathway数据库分析靶基因所参与的骨化相关信号传导通路;使用TRANSFAC7.0public转录因子数据库及Patser预测程序预测转录因子结合位点。结果成纤维细胞在HAAM上生长时形态保持良好,呈簇状分布,复层生长并建立起细胞间联系;免疫荧光鉴定发现DISH组细胞骨钙素和Ⅰ、Ⅱ、Ⅲ型胶原呈阳性,正常组细胞Ⅰ和Ⅲ型胶原呈阳性;共获得15种信号比值〉1.5倍差异表达的miRNAs,12种上调、3种下调,qRT-PCR验证结果与微阵列芯片检测结果一致;共预测出67个靶基因,影响细胞分化、黏附及矿物质沉积等活动,参与MAPK、Wnt、TGF-β、Focaladhesion等多个骨化相关信号途径,预测出10种差异性miRNAs的转录因子。结论HAAM可实现成纤维细胞在体外三维黏附下生长,部分miRNAs的异常表达可能参与DISH发病。
Objective To obtain and anlysis the diffuse idiopathic skeletal hyperostosis(DISH) related miRNAs under 3- D adhesion for cell culture. Methods From January 2012 to January 2014, 4 ossific ligamenta flava tissues were obtained from DISH patients and 4 normal ligamenta flava tissues were obtained from trauma patients surgically. Fibroblasts were separated by using collagenase technique and then cultured on human acellular amniotic membrane (HAAM). Each sample was identified by immunofluorescence before harvested. Total RNA was extracted and then quantified by microfluidics analysis. The small RNAs (〈 300 nt) were isolated by using a YM-100 Microeon centrifugal filter, μParafloTM MiRNA microarray assay was performed using a service provider to identify miRNAs whose expression was significantly different between the two groups. Part of differential expression miRNAs were verified by qRT-PCR. Targets of miRNAs were obtained using PicTar 2005, miRanda vS, TargetScan 5.1, their function were analyzed by using Gene Ontology. Functional pathway analysis of miRNAs was performed using KEGG Path- way Analysis. TRANSFAC 7.0 public and Parser were used to get the distribution of transcription factor binding sites. Results When grown on HAAM, fibroblasts kept their morphology, distributed in the way of cluster, lived in multi-level of HAAM, and es- tablished linkage. Collagen Ⅰand Ⅲ were tested positive in normal group ceils. Collagen Ⅰ, Ⅱ, Ⅲ and Osteocalcin were tested posi- tive in DISH group cells by immunefluorescence. In total 15 miRNAs showed differential expression, 12 were up-regulated and 3 were down-regulated. The result of qRT-PCR was consistent with MiRNA microarray assay. Totally 67 target genes were predicted which participated in cell differentiation, cell adhesion, mineralization et al, and had function in regulating MAPK, Wnt, TGF-β, Focal adhesion signal pathway et al. In total 10 transcription factors were predicted in differentially expressed miRNAs. Conclu- sion HAAM can provide fibroblasts with 3D adhesion growth, Some differentially expressed miRNAs may participate in the oathogenesis of DISH.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2015年第1期68-75,共8页
Chinese Journal of Orthopaedics
基金
国家自然科学基金项目(81271360,81471403)
关键词
微RNAS
黄韧带
骨化
异位性
微阵列分析
MicroRNAs
Ligamentum flavum, Ossification, heterotopic
Microarray analysis