CAJ | 학술논문

目的探讨高糖刺激内皮细胞表型改变后是否诱导其分化为多能干细胞,再进一步分化成软骨细胞,从而介导血管钙化的形成.方法人主动脉内皮细胞(HAEC)被随机分成3组:正常对照组(NG,5.5 mmol/L葡萄糖)、高糖组(HG,30 mmol/L葡萄糖)和甘露醇对照组(5.5 mmol/L葡萄糖+24.5 mmol/L甘露醇),培养48 h.激光共聚焦显微镜观察CD31和成纤维细胞特异性蛋白1(FSP1)在人主动脉内皮细胞的表达.实时定量PCR和Western印迹法检测细胞CD31及FSP1基因和蛋白的表达.再使用软骨细胞培养基对高糖刺激的人主动脉内皮细胞进行1周诱导分化,实时定量PCR和Western印迹法检测细胞CD10和CD44基因和蛋白的表达,免疫荧光法检测间充质干细胞标志物CD44和STRO-1的表达；阿辛蓝染色和Western印迹法鉴定软骨细胞和其标志物SOX9的表达.电镜观察细胞形态变化.结果高糖干预后,CD31的蛋白和mRNA表达水平下降(P＜0.01),FSP1的蛋白和mRNA表达水平增加(P＜0.01)；激光共聚焦显微镜可见CD31和FSP1表达重叠,一些细胞呈纺锤样改变并CD31染色阴性.经软骨细胞培养基诱导分化1周后,多能干细胞标志物CD44、CD10的蛋白和mRNA表达水平显著高于正常对照组(P＜ 0.01)；共聚焦显微镜提示,间充质干细胞标志物STRO-1表达亦显著高于正常对照组；高糖组细胞SOX9蛋白和基因表达水平显著高于正常对照组(P＜0.01)；细胞阿辛蓝和茜素红染色阳性；电镜显示高糖使细胞粗面内质网和微丝明显增多.结论高糖可诱导HAEC发生内皮-间充质转分化,并获得多能干细胞表型,在合适的诱导环境下分化为软骨细胞,参与糖尿病肾病心血管钙化的发生和发展.
목적 탐토고당자격내피세포표형개변후시부유도기분화위다능간세포,재진일보분화성연골세포,종이개도혈관개화적형성.방법 인주동맥내피세포(HAEC)피수궤분성3조:정상대조조(NG,5.5 mmol/L포도당)、고당조(HG,30 mmol/L포도당)화감로순대조조(5.5 mmol/L포도당+24.5 mmol/L감로순),배양48 h.격광공취초현미경관찰CD31화성섬유세포특이성단백1(FSP1)재인주동맥내피세포적표체.실시정량PCR화Western인적법검측세포CD31급FSP1기인화단백적표체.재사용연골세포배양기대고당자격적인주동맥내피세포진행1주유도분화,실시정량PCR화Western인적법검측세포CD10화CD44기인화단백적표체,면역형광법검측간충질간세포표지물CD44화STRO-1적표체；아신람염색화Western인적법감정연골세포화기표지물SOX9적표체.전경관찰세포형태변화.결과 고당간예후,CD31적단백화mRNA표체수평하강(P＜0.01),FSP1적단백화mRNA표체수평증가(P＜0.01)；격광공취초현미경가견CD31화FSP1표체중첩,일사세포정방추양개변병CD31염색음성.경연골세포배양기유도분화1주후,다능간세포표지물CD44、CD10적단백화mRNA표체수평현저고우정상대조조(P＜ 0.01)；공취초현미경제시,간충질간세포표지물STRO-1표체역현저고우정상대조조；고당조세포SOX9단백화기인표체수평현저고우정상대조조(P＜0.01)；세포아신람화천소홍염색양성；전경현시고당사세포조면내질망화미사명현증다.결론 고당가유도HAEC발생내피-간충질전분화,병획득다능간세포표형,재합괄적유도배경하분화위연골세포,삼여당뇨병신병심혈관개화적발생화발전.
Objective To explore whether high glucose (HG)-induced endothelial-to-mesenchymal transition (EndMT) could be transitioned into mesenchymal stem cells (MSCs) and further differentiated into chondrocytes.Methods Human aortic endothelial cells (HAECs) were divided into three groups:normal glucose (NG,5.5 mmol/L glucose) group,HG (30 mmol/L glucose) group,and mannitol (5.5 mmol/L glucose + 24.5 mmol/L mannitol) group,and were cultured for 48 h.Immunofluorescence staining was performed to detect the co-expression of CD31 (endothelial markers),and fibroblast-specific protein 1 (FSP1,fibroblast markers).The expression of CD31 and FSP1 mRNA and protein was detected by real-time PCR and Western blotting.When endothelial-derived MSCs were grown in MSC medium for one week,the expression of the MSCs markers CD44,CD10 and the chondrocyte marker SOX9 was detected by Western blotting and RT-PCR.Chondrocyte expression was detected by alcian blue staining.Calcium deposit was analyzed by alizarin red staining.Pathological changes were investigated using electron microscopy.Results The expression of FSP1 mRNA and protein was significantly increased,but the expression of CD31 mRNA and protein was decreased (P ＜0.01),and the cells undergoing EndMT also significantly expressed CD10,CD44 and SOX9 in the HG group compared with those in normal glucose group (P ＜ 0.01).The incubation of HAECs exposed to HG resulted in a fibroblast-like phenotype,wherein increased microfilamentation and a roughened endoplasmic reticulum structure were observed in the cytoplasm.Double staining of the HAECs indicated a co-localization of CD31 and FSP1.After one week culture for chondrocyte medium,the expression of MSCs marker STRO-1 was significantly increased by immunofluorescence staining.Additionally,aleian blue staining in the HG group was positive compared to the NG group.Consistent with the elevation of SOX9 expression,calcium deposit also enhanced in the HG group.Conclusion HG can induce endothelial cells transdifferentiation into chondrocyte-like cells via the EndMT.