CAJ | 학술논문

目的探讨整合素连接激酶(intergrin-linked kinase,ILK)对增生性瘢痕血管生成的影响及调控机制.方法收集6例增生期的瘢痕组织标本,体外分离培养瘢痕微血管内皮细胞(HSMECs),取2～4代生长状态良好的细胞,并分成4组:空白对照组,常规细胞培养;阴性对照组,只加入Lipo2000转染试剂;LY294002组,加入终浓度50 nmol/L的LY294002;ILK siRNA组,加入终浓度为20 nmol/L的ILK siRNA.转染48 h后,采用RT-PCR法及Western Blot法检测ILK mRNA和蛋白表达的变化.利用Transwell法,收集转染后的4组HSMECs,用不含血清的DMEM重悬,种入上层小室,下层加完全培养基,10h后终止实验,计算各组细胞迁移数,观察不同处理因素对HSMECs迁移能力的影响.将冻融的ECMatrix基质胶在冰面上均匀的铺入预冷的48孔板中,37℃温箱孵育使其凝胶,然后取转染后的4组细胞种入胶面上,8h后终止实验,依据血管形成模式评分表随机选择8个视野进行评分,分析各组细胞的体外血管形成能力.结果 ①与空白对照组(ILK mRNA=0.829±0.109、ILK蛋白=1)相对比,ILK siRNA组HSMECs内ILK mRNA表达水平(ILK mRNA=0.002±0.000;t=13.151,P=0.006)及蛋白表达水平(ILK蛋白=0.096±0.049;t=36.656,P=0.000)均明显受到抑制,而LY294002组ILK的表达虽略有降低,但差异无统计学意义.②ILK siRNA组与LY294002组在10 h时的细胞迁移数分别为88.111 ±3.079、138.667±2.404,较空白对照组322.333±3.712显著降低(P＜0.05).③在体外血管形成8h时,ILK siRNA组(2.625±0.125)和LY294002组(3.125±0.250)与空白对照组(4.333±0.191)相比,体外血管形成受到明显的抑制,无法形成完整的复杂网络结构(P＜0.05).结论在ILK表达下调的情况下,HSMECs的迁移能力与体外血管形成能力均受到明显抑制,推论ILK可能参与对瘢痕血管生成的调控.
목적 탐토정합소련접격매(intergrin-linked kinase,ILK)대증생성반흔혈관생성적영향급조공궤제.방법 수집6례증생기적반흔조직표본,체외분리배양반흔미혈관내피세포(HSMECs),취2～4대생장상태량호적세포,병분성4조:공백대조조,상규세포배양;음성대조조,지가입Lipo2000전염시제;LY294002조,가입종농도50 nmol/L적LY294002;ILK siRNA조,가입종농도위20 nmol/L적ILK siRNA.전염48 h후,채용RT-PCR법급Western Blot법검측ILK mRNA화단백표체적변화.이용Transwell법,수집전염후적4조HSMECs,용불함혈청적DMEM중현,충입상층소실,하층가완전배양기,10h후종지실험,계산각조세포천이수,관찰불동처리인소대HSMECs천이능력적영향.장동융적ECMatrix기질효재빙면상균균적포입예랭적48공판중,37℃온상부육사기응효,연후취전염후적4조세포충입효면상,8h후종지실험,의거혈관형성모식평분표수궤선택8개시야진행평분,분석각조세포적체외혈관형성능력.결과 ①여공백대조조(ILK mRNA=0.829±0.109、ILK단백=1)상대비,ILK siRNA조HSMECs내ILK mRNA표체수평(ILK mRNA=0.002±0.000;t=13.151,P=0.006)급단백표체수평(ILK단백=0.096±0.049;t=36.656,P=0.000)균명현수도억제,이LY294002조ILK적표체수략유강저,단차이무통계학의의.②ILK siRNA조여LY294002조재10 h시적세포천이수분별위88.111 ±3.079、138.667±2.404,교공백대조조322.333±3.712현저강저(P＜0.05).③재체외혈관형성8h시,ILK siRNA조(2.625±0.125)화LY294002조(3.125±0.250)여공백대조조(4.333±0.191)상비,체외혈관형성수도명현적억제,무법형성완정적복잡망락결구(P＜0.05).결론 재ILK표체하조적정황하,HSMECs적천이능력여체외혈관형성능력균수도명현억제,추론ILK가능삼여대반흔혈관생성적조공.
Objective To investigate the effects and regulatory mechanism of ILK on angiogenesis in hypertrophic scar.Methods The human scar microvascular endothelial cells (HSMECs) were isolated from 6 patients' hypertrophic scar in vitro.The HSMECs with good condition in 2nd to 4th generation were selected as experimental objectives.①HSMECs were divided into the blank control group (treated with routine culture),negative control group (treated with only Lipofectamine 2000),LY294002 group (incubated with 50 nmol/L LY294002),ILK siRNA group (incubated with 20 nmol/L ILK siRNA).RTPCR and Western Blot were used to detect the expression of ILK mRNA and its protein after transfecion for 48 h.② The digested HSMECs of four groups were resuspended with DMEM without serum and then seeded onto the upper compartment of transwell insert which contained complete medium in its lower compartment.The cell migration experiment was stopped in 10 h and then the migrated cells were counted to analyze the effects of different interventions on the migration ability of HSMECs.③ The thawed ECMatrixTM was put into each well of pre-colled 48-well tissue culture plate,and then the plate was put into the incubator at 37 ℃ to make it to become gel.The HSMECs of four groups were seeded onto the surface of the ECMatrix gel and were put into incubator.Eight random view-fields per well should be valued by the sheet of pattern recognition about angiogenesis after 8 hours to evaluate the ability of angiogenesis in vitro between four groups.Results ① The expression of ILK mRNA (ILK mRNA =0.829 ± 0.109,t =13.151,P=0.006) and protein (ILK protein =0.096 ±0.049,t =36.656,P =0.000) were both inhibited obviously in ILK siRNA group compared with the blank control group (ILK mRNA =0.829 ±0.109,ILK protein =1).And,the expression of ILK in LY294002 group was slightly lower than that of black control group,but there was no statistical difference.② The number of migrated cells in ILK siRNA group (88.111 ± 3.079) and LY294002 group (138.667 ± 2.404) were respectively lower than that in blank control group (322.333 ± 3.712,P ＜ 0.05) in 10th hour.③ Compared to blank control group (4.333 ±0.191),the ability of angiogenesis in vitro decreased significantly ILK siRNA group (2.625 ±0.125) and LY294002 group (3.125 ± 0.250),in which,the vascular network structures were not formed perfectly in 8th hour (P ＜0.05).Conclusions The ability of HSMECs' migration and angiogenesis in vitro are inhibited significantly when the expression of ILK is down-regulated.It reveals that ILK may play an role in the regulation of scar angiogenesis.