人牙髓细胞内皮向分化中 miRNA 的表达及生物信息学分析  

Expression profiles and bioinformatic analysis of miRNA in human dental pulp cells during endothelial differentiation

作　　者：龚启梅[1] 蒋宏伟[1] 王劲茗[2] 凌均棨[1] 

机构地区：[1]中山大学光华口腔医学院·附属口腔医院牙体牙髓病科· 广东省口腔医学重点实验室,广州510055 [2]中山大学光华口腔医学院·种植科,广州510055

出　　处：《中华口腔医学杂志》2014年第5期284-289,共6页Chinese Journal of Stomatology

基　　金：国家自然科学基金（81200775）;广东省医学科研基金（B2012140）

摘　　要：目的研究人牙髓细胞内皮向分化时差异表达的微小RNA（ microRNA，miRNA）并进行生物信息学分析，初步探讨miRNA在人牙髓细胞内皮向分化中的作用。方法以添加50μg/L血管内皮生长因子（vascular endothelial growth factor，VEGF）、10μg/L碱性成纤维细胞生长因子（basic fibroblast growth factor ，bFGF）和2％胎牛血清的培养基培养人牙髓细胞7 d为诱导组，以未诱导的人牙髓细胞为对照组，实时荧光定量PCR检测血管内皮标志基因血小板-内皮细胞黏附分子（ CD31）、冯·维勒布兰德因子（ von Willebrand factor ， vWF ）和血管内皮细胞钙黏着蛋白（ vascular endothelial-cadherin，VE-cad）的表达；体外成管实验检测诱导后细胞的成管能力；通过miRNA表达谱芯片检测miRNA的表达并采用实时荧光定量PCR进行验证；运用生物信息学软件预测差异表达miRNA调节的靶基因及可能涉及的生物学功能和信号通路。结果实时荧光定量PCR结果显示，CD31、vWF和VE-cad在对照组牙髓细胞中的相对表达量分别为（3.48±0.22）×10^-4、（3.13±0.31）×10^-4和（39.60±2.36）×10^-4，而在诱导组的相对表达量则分别为（19.57±2.20）×10^-4、（48.13±0.54）×10^-4及（228.00±8.89）×10^-4，3种基因在诱导组的表达均较对照组显著上调（P＜0.05）；成管实验显示，诱导后细胞在基质胶上可形成类似小管样结构；基因芯片结果显示，人牙髓细胞内皮向诱导后差异表达的miRNA有47个，其中15个miRNA表达上调，32个miRNA表达下调；实时荧光定量PCR对上述部分差异表达miRNA的验证结果与芯片结果基本一致；生物信息学分析显示，上述差异表达miRNA的靶基因显著富集于转录调控、细胞运动、血管形态、血管新生和细胞骨架蛋白等生物学功能，且与丝裂原活化蛋白激酶通路和Wnt通路等信号通路有关。结论人牙髓细胞内皮向诱导后存在miRNA的差异表Objective To investigate the differential expression profile and bioinformatic analysis of microRNA（miRNA） in human dental pulp cells （DPC） during endothelial differentiation.Methods DPC were cultured in endothelial induction medium （ 50 μg/L vascular endothelial growth factor ,10 μg/L basic fibroblast growth factor and 2% fetal calf serum ） for 7 days.Meanwhile non-induced DPC were used as control.Quantitative real-time PCR （ qRT-PCR ） was applied to detect vascular endothelial marker genes [CD31,von Willebrand factor（vWF） and vascular endothelial-cadherin（VE-cadherin）] and in vitro tube formation on matrigel was used to analyze the angiogenic ability of differentiated cells.And then miRNA expression profiles of DPC were examined using miRNA microarray and then the differentially expressed miRNA were validated by qRT-PCR.Furthermore,bioinformatic analysis was employed to predict the target genes of miRNA and to analyze the possible biological functions and signaling pathways that were involved in DPC after induction.Results The relative mRNA level of CD 31 ,vWF and VE-cadherin in the control group＆amp;nbsp;were （3.48 ±0.22） ×10^-4 ,（3.13 ±0.31） ×10^-4 and （39.60 ±2.36） ×10^-4 ,and （19.57 ±2.20） × 10^-4 ,（48.13 ±0.54） ×10^-4 and （228.00 ±8.89） ×10^-4 in the induced group.The expressions of CD31 , vWF and VE-cadherin were increased significantly in endothelial induced DPC compared to the control group （P＆lt;0.05）.For in vitro tube formation assay,tubular structures were formed on the matrigel by differentiated DPC.A total of 47 miRNA were differentially expressed , in which 15 miRNA were up-regulated and 32 miRNAs down-regulated in differentiated DPC compared with the control.Of these,4 miRNA were confirmed by qRT-PCR.The target genes of differential miRNA were predicted to associate with several biological functions,such as the regulation of transcription ,cell motion,blood vessel morphogenesis ,angiogenesis and cytoskeletal protein ,and signal

关 键 词：细胞分化 计算生物学 牙髓细胞 微小RNA 血管新生 

分 类 号：R780.2[医药卫生—口腔医学]