CAJ | 학술논문

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的差异表达，为牙髓细胞的分化机制和牙髓血管新生提供新的思路。目的研究人牙髓細胞內皮嚮分化時差異錶達的微小RNA（ microRNA，miRNA）併進行生物信息學分析，初步探討miRNA在人牙髓細胞內皮嚮分化中的作用。方法以添加50μg/L血管內皮生長因子（vascular endothelial growth factor，VEGF）、10μg/L堿性成纖維細胞生長因子（basic fibroblast growth factor ，bFGF）和2％胎牛血清的培養基培養人牙髓細胞7 d為誘導組，以未誘導的人牙髓細胞為對照組，實時熒光定量PCR檢測血管內皮標誌基因血小闆-內皮細胞黏附分子（ CD31）、馮·維勒佈蘭德因子（ von Willebrand factor ， vWF ）和血管內皮細胞鈣黏著蛋白（ vascular endothelial-cadherin，VE-cad）的錶達；體外成管實驗檢測誘導後細胞的成管能力；通過miRNA錶達譜芯片檢測miRNA的錶達併採用實時熒光定量PCR進行驗證；運用生物信息學軟件預測差異錶達miRNA調節的靶基因及可能涉及的生物學功能和信號通路。結果實時熒光定量PCR結果顯示，CD31、vWF和VE-cad在對照組牙髓細胞中的相對錶達量分彆為（3.48±0.22）×10-4、（3.13±0.31）×10-4和（39.60±2.36）×10-4，而在誘導組的相對錶達量則分彆為（19.57±2.20）×10-4、（48.13±0.54）×10-4及（228.00±8.89）×10-4，3種基因在誘導組的錶達均較對照組顯著上調（P＜0.05）；成管實驗顯示，誘導後細胞在基質膠上可形成類似小管樣結構；基因芯片結果顯示，人牙髓細胞內皮嚮誘導後差異錶達的miRNA有47箇，其中15箇miRNA錶達上調，32箇miRNA錶達下調；實時熒光定量PCR對上述部分差異錶達miRNA的驗證結果與芯片結果基本一緻；生物信息學分析顯示，上述差異錶達miRNA的靶基因顯著富集于轉錄調控、細胞運動、血管形態、血管新生和細胞骨架蛋白等生物學功能，且與絲裂原活化蛋白激酶通路和Wnt通路等信號通路有關。結論人牙髓細胞內皮嚮誘導後存在miRNA的差異錶達，為牙髓細胞的分化機製和牙髓血管新生提供新的思路。
목적연구인아수세포내피향분화시차이표체적미소RNA（ microRNA，miRNA）병진행생물신식학분석，초보탐토miRNA재인아수세포내피향분화중적작용。방법이첨가50μg/L혈관내피생장인자（vascular endothelial growth factor，VEGF）、10μg/L감성성섬유세포생장인자（basic fibroblast growth factor ，bFGF）화2％태우혈청적배양기배양인아수세포7 d위유도조，이미유도적인아수세포위대조조，실시형광정량PCR검측혈관내피표지기인혈소판-내피세포점부분자（ CD31）、풍·유륵포란덕인자（ von Willebrand factor ， vWF ）화혈관내피세포개점착단백（ vascular endothelial-cadherin，VE-cad）적표체；체외성관실험검측유도후세포적성관능력；통과miRNA표체보심편검측miRNA적표체병채용실시형광정량PCR진행험증；운용생물신식학연건예측차이표체miRNA조절적파기인급가능섭급적생물학공능화신호통로。결과실시형광정량PCR결과현시，CD31、vWF화VE-cad재대조조아수세포중적상대표체량분별위（3.48±0.22）×10-4、（3.13±0.31）×10-4화（39.60±2.36）×10-4，이재유도조적상대표체량칙분별위（19.57±2.20）×10-4、（48.13±0.54）×10-4급（228.00±8.89）×10-4，3충기인재유도조적표체균교대조조현저상조（P＜0.05）；성관실험현시，유도후세포재기질효상가형성유사소관양결구；기인심편결과현시，인아수세포내피향유도후차이표체적miRNA유47개，기중15개miRNA표체상조，32개miRNA표체하조；실시형광정량PCR대상술부분차이표체miRNA적험증결과여심편결과기본일치；생물신식학분석현시，상술차이표체miRNA적파기인현저부집우전록조공、세포운동、혈관형태、혈관신생화세포골가단백등생물학공능，차여사렬원활화단백격매통로화Wnt통로등신호통로유관。결론인아수세포내피향유도후존재miRNA적차이표체，위아수세포적분화궤제화아수혈관신생제공신적사로。
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 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<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 signaling pathways including the mitogen-activated protein kinase ( MAPK) and the Wnt signaling pathway.Conclusions The differential miRNA expression identified in this study may be involved in governing DPC endothelial differentiation , thus contributing to the future research on regulatory mechanisms in dental pulp angiogenesis.