QLT0267抑制整合素连接激酶对人皮肤成纤维细胞生物学特征的影响  

Effects of different concentrations of ILK inhibitor QLT0267 on biological characteristics of human skin fibroblast cells

作　　者：梁振文[1] 李叶扬[2] 林伟华[2] 李罡[2] 孙敬恩[2] 王仁坤[3] 王晓红[2] 

机构地区：[1]暨南大学附属第一医院东圃分院康复科,广州510660 [2]暨南大学附属广州市红十字会医院烧伤整形科,510220 [3]合肥市第一人民医院滨湖医院烧伤整形科,230601

出　　处：《中华损伤与修复杂志（电子版）》2013年第6期18-22,共5页Chinese Journal of Injury Repair and Wound Healing(Electronic Edition)

基　　金：广东省自然科学基金项目(S2011010000764);广州市医药卫生科技重点项目(2009-ZPi-05);广东省社会发展领域科技计划项目(2010-75)

摘　　要：目的观察不同浓度的QLT0267抑制整合素连接激酶(ILK)的活性对成纤维细胞(Fb)生物学特征的影响。方法收集整形手术后剩余的皮肤标本,采用机械法加酶消化法分离培养Fb,随机分为空白对照组、5 nM组、10 nM组、20 nM组、30 nM组、DMSO组,其中空白对照组常规培养不做任何处理,5 nM组、10 nM组、20 nM组、30 nM组按培养基中QLT0267浓度分别为5、10、20、30 nmol/L加入QLT0267溶液[二甲基亚酮(DMSO)为溶剂],DMSO组加入与30 nM组中QLT0267溶液同体积的DMSO,分组处理12、24、48、72 h后在倒置相差显微镜下观察细胞形态。CellTiter试剂盒检测分组处理24、48、72 h后各组Fb的增殖情况,并与空白对照组比较计算增殖抑制率;在48 h时间点,流式细胞仪检测各组细胞凋亡情况以及Western Blot法观察Fb表达磷酸化蛋白激酶B(pAKT)、总蛋白激酶B(tAKT)蛋白的变化。对数据进行LDS-t检验和重复测量设计方差分析检验。结果经浓度大于10 nmol/L的ILK特异性抑制剂作用下,Fb膨胀,细胞核固缩或破碎,并出现细胞凋亡,随QLT0267浓度的增高和刺激时间的延长,细胞形态的改变更加明显。CellTiter试剂盒检测结果显示10 nmol/L以上的QLT0267能明显抑制Fb的增殖,且随着QLT0267浓度的增高和刺激时间的延长,增殖抑制率也升高(F处理组=94.242,P<0.05;F时间=240.490,P<0.05;F处理与时间=11.551,P<0.05)。流式细胞仪检测结果显示与对照组比较,10 nmol/L以上的ILK特异性抑制剂能诱导Fb凋亡,差异有统计学意义(P<0.05)。Western Blot结果显示在各组tAKT的表达组间差异无统计学意义(P>0.05)的前提下,不同浓度QLT0267组pAKT的表达比空白对照组低,并随着QLT0267浓度的上升而pAKT蛋白生成下降(P<0.05)。结论 10 nmol/L浓度以上ILK特异性抑制剂QLT0267能刺激Fb发生形态改变,抑制细胞增殖和促进细胞凋亡。推测ILK可能通过影响Fb中pAKT的生成参与创面愈合过程的调控。Objective To investigate the biological changes of fibroblast( Fb) by using different concentrations of integrin-linked kinase( ILK) inhibitor QLT0267 in different time points. Methods Skin Fb were isolated from redundant grafts of plastic surgery patients by mechanical method combined with enzyme digestion. The experiments were randomly divided into 6 groups: blank control group,5 nM group, 10 nM group,20 nM group,30 nM group,DMSO group. No stimulation factor was added in the blank control group,5 nM,10 nM,20 nM,30 nM groups were added with QLT0267 solution according to the concentrations of QLT0267 in the culture medium as 5,10,20,30( nmol / L). DMSO group was added with DMSO in the same volume as the QLT0267 solution of 30 nm group. Firstly the cell morphology under inverted phase contrast microscope was observed at 12,24,48,72 h. And then cell proliferation was detected at 24,48,72h by CellTiter kit. Finally the flow cytometry and Western Blot detection were used to detect the apoptosis of cells and pAKT,tAKT protein expression of the cell at 48h. Data analysis was conducted by using LDS-t and repeated measurement ANOVA to determine significance between samples. Results More than 10 nnmol /L inhibitors of ILK could make Fb swell and expand roundly,followed by the nuclear pyknosis or disintegration,even the apoptosis appeared. It was more obvious with longer time of drug stimulation and higher concentration of drug. The results of CellTiter kit showed that the ILK specific inhibitor QLT0267 with 10 nmol / L and more could inhibit the cell proliferation( P < 0. 05),and as the concentration increased and the time for stimulation extended the inhibition rate of proliferation would ascensus( Fgroup = 94. 242,P < 0. 05; Ftime = 240. 490,P < 0. 05; Fgroup&time = 11. 551,P < 0. 05). The results of flow cytometry suggested that the ILK specific inhibitor QLT0267 with 10 nmol / L and more could significantly induce Fb apoptosis than the control group( P < 0. 05). But Western Blot displayed that there was no expression

关 键 词：伤口愈合 成纤维细胞 整合素连接激酶 QLT0267 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]