中华整形外科杂志
中華整形外科雜誌
중화정형외과잡지
CHINESE JOURNAL OF PLASTIC SURGERY
2014年
1期
45-49
,共5页
林伟华%李叶扬%米兰%李罡%孙敬恩%王仁坤%梁振文
林偉華%李葉颺%米蘭%李罡%孫敬恩%王仁坤%樑振文
림위화%리협양%미란%리강%손경은%왕인곤%량진문
整合素连接激酶%瘢痕%成纤维细胞%α-平滑肌肌动蛋白
整閤素連接激酶%瘢痕%成纖維細胞%α-平滑肌肌動蛋白
정합소련접격매%반흔%성섬유세포%α-평활기기동단백
Integrin-linked kinase%Cicatrix%Fibroblast%α-smooth muscle actin
目的 观察整合素连接激酶(integrin-linked kinase,ILK)对人瘢痕成纤维细胞增殖和分化的影响,以探讨其在瘢痕形成中的作用.方法 体外分离培养瘢痕成纤维细胞,并按下述方法处理并分组:①空白对照组,仅含有常规高糖DMEM培养液;②转染试剂jetPRIMETM对照组(jetPRIMETM对照组),常规高糖DMEM培养液与200μl jetPRIMETM buffer及4μl jetPRIMETM反应试剂;③ILK小分子干扰RNA(small interfering RNA,siRNA)组(ILK siRNA组),常规高糖DMEM培养液与siRNA转染复合物;④ILK互补DNA(complementary DNA,cDNA)组(ILK cDNA组),常规高糖DMEM培养液与cDNA转染复合物.分别检测细胞增殖、ILK信使RNA(messenger RNA,mRNA)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)mRNA表达及其蛋白表达水平.结果 ①四唑鎓盐(XTT)检测结果表明,转染后48 h,各组成纤维细胞增殖水平,空白对照组为0.820±0.065,jetPRIMETM对照组为0.873±0.041,ILK siRNA组为0.554±0.013,ILK cDNA组为1.296±0.094.各组48 h内的细胞增殖曲线见,ILK siRNA组的细胞增殖极为平缓,而ILK cDNA组的细胞增殖水平从12 h开始逐渐升高.转染后48 h,ILK siRNA组细胞增殖水平明显低于其他3组(P值分别为0.021、0.034、0),ILK cDNA组细胞增殖水平明显高于其他3组(P值分别为0.017、0.009、0).②实时荧光定量聚合酶链式反应(Real-time qPCR)技术检测结果显示,ILK mRNA和α-SMA mRNA的表达水平,空白对照组为0.693±0.412和0.422±0.037,jetPRIMETM对照组为0.621±0.183和0.388±0.005,ILK siRNA组为0.052±0.019和0.073±0.023,ILK cDNA组为240.193±35.170和138.056±24.060.ILK siRNA组ILK mRNA和α-SMA mRNA表达水平均显著低于其他组,差异有统计学意义(P＜0.05);而ILK cDNA组ILK mRNA和α-SMA mRNA表达水平均显著高于其他3组,差异有统计学意义(P＜0.05).③应用Western blot检测发现ILK siRNA组的ILK及α-SMA蛋白表达明显受到抑制,而ILK cDNA组的ILK及α-SMA蛋白表达均明显增加.结论 ILK对成纤维细胞增殖具有促进作用,并能够在转录和翻译水平对α-SMA进行上调控制,可能通过促进成纤维细胞向肌成纤维细胞的分化,并在瘢痕的形成与挛缩中起到重要作用.
目的 觀察整閤素連接激酶(integrin-linked kinase,ILK)對人瘢痕成纖維細胞增殖和分化的影響,以探討其在瘢痕形成中的作用.方法 體外分離培養瘢痕成纖維細胞,併按下述方法處理併分組:①空白對照組,僅含有常規高糖DMEM培養液;②轉染試劑jetPRIMETM對照組(jetPRIMETM對照組),常規高糖DMEM培養液與200μl jetPRIMETM buffer及4μl jetPRIMETM反應試劑;③ILK小分子榦擾RNA(small interfering RNA,siRNA)組(ILK siRNA組),常規高糖DMEM培養液與siRNA轉染複閤物;④ILK互補DNA(complementary DNA,cDNA)組(ILK cDNA組),常規高糖DMEM培養液與cDNA轉染複閤物.分彆檢測細胞增殖、ILK信使RNA(messenger RNA,mRNA)、α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)mRNA錶達及其蛋白錶達水平.結果 ①四唑鎓鹽(XTT)檢測結果錶明,轉染後48 h,各組成纖維細胞增殖水平,空白對照組為0.820±0.065,jetPRIMETM對照組為0.873±0.041,ILK siRNA組為0.554±0.013,ILK cDNA組為1.296±0.094.各組48 h內的細胞增殖麯線見,ILK siRNA組的細胞增殖極為平緩,而ILK cDNA組的細胞增殖水平從12 h開始逐漸升高.轉染後48 h,ILK siRNA組細胞增殖水平明顯低于其他3組(P值分彆為0.021、0.034、0),ILK cDNA組細胞增殖水平明顯高于其他3組(P值分彆為0.017、0.009、0).②實時熒光定量聚閤酶鏈式反應(Real-time qPCR)技術檢測結果顯示,ILK mRNA和α-SMA mRNA的錶達水平,空白對照組為0.693±0.412和0.422±0.037,jetPRIMETM對照組為0.621±0.183和0.388±0.005,ILK siRNA組為0.052±0.019和0.073±0.023,ILK cDNA組為240.193±35.170和138.056±24.060.ILK siRNA組ILK mRNA和α-SMA mRNA錶達水平均顯著低于其他組,差異有統計學意義(P＜0.05);而ILK cDNA組ILK mRNA和α-SMA mRNA錶達水平均顯著高于其他3組,差異有統計學意義(P＜0.05).③應用Western blot檢測髮現ILK siRNA組的ILK及α-SMA蛋白錶達明顯受到抑製,而ILK cDNA組的ILK及α-SMA蛋白錶達均明顯增加.結論 ILK對成纖維細胞增殖具有促進作用,併能夠在轉錄和翻譯水平對α-SMA進行上調控製,可能通過促進成纖維細胞嚮肌成纖維細胞的分化,併在瘢痕的形成與攣縮中起到重要作用.
목적 관찰정합소련접격매(integrin-linked kinase,ILK)대인반흔성섬유세포증식화분화적영향,이탐토기재반흔형성중적작용.방법 체외분리배양반흔성섬유세포,병안하술방법처리병분조:①공백대조조,부함유상규고당DMEM배양액;②전염시제jetPRIMETM대조조(jetPRIMETM대조조),상규고당DMEM배양액여200μl jetPRIMETM buffer급4μl jetPRIMETM반응시제;③ILK소분자간우RNA(small interfering RNA,siRNA)조(ILK siRNA조),상규고당DMEM배양액여siRNA전염복합물;④ILK호보DNA(complementary DNA,cDNA)조(ILK cDNA조),상규고당DMEM배양액여cDNA전염복합물.분별검측세포증식、ILK신사RNA(messenger RNA,mRNA)、α-평활기기동단백(α-smooth muscle actin,α-SMA)mRNA표체급기단백표체수평.결과 ①사서옹염(XTT)검측결과표명,전염후48 h,각조성섬유세포증식수평,공백대조조위0.820±0.065,jetPRIMETM대조조위0.873±0.041,ILK siRNA조위0.554±0.013,ILK cDNA조위1.296±0.094.각조48 h내적세포증식곡선견,ILK siRNA조적세포증식겁위평완,이ILK cDNA조적세포증식수평종12 h개시축점승고.전염후48 h,ILK siRNA조세포증식수평명현저우기타3조(P치분별위0.021、0.034、0),ILK cDNA조세포증식수평명현고우기타3조(P치분별위0.017、0.009、0).②실시형광정량취합매련식반응(Real-time qPCR)기술검측결과현시,ILK mRNA화α-SMA mRNA적표체수평,공백대조조위0.693±0.412화0.422±0.037,jetPRIMETM대조조위0.621±0.183화0.388±0.005,ILK siRNA조위0.052±0.019화0.073±0.023,ILK cDNA조위240.193±35.170화138.056±24.060.ILK siRNA조ILK mRNA화α-SMA mRNA표체수평균현저저우기타조,차이유통계학의의(P＜0.05);이ILK cDNA조ILK mRNA화α-SMA mRNA표체수평균현저고우기타3조,차이유통계학의의(P＜0.05).③응용Western blot검측발현ILK siRNA조적ILK급α-SMA단백표체명현수도억제,이ILK cDNA조적ILK급α-SMA단백표체균명현증가.결론 ILK대성섬유세포증식구유촉진작용,병능구재전록화번역수평대α-SMA진행상조공제,가능통과촉진성섬유세포향기성섬유세포적분화,병재반흔적형성여련축중기도중요작용.
Objective To study the role of integrin-linked kinase (ILK)on the proliferation and differentiation of human fibroblast in hypertrophic scar and its effect on the scar formation.Methods The human scar fibroblasts were isolated and cultured in vitro.The cells were divided into 4 groups.①control group:only contains DMEM; ②jetPRIMETM group:DMEM with 200 μl jetPRIMETM buffer and 4 μl jetPRIMETM ; ③ILK siRNA group:DMEM and ILK siRNA; ④ILK cDNA group:DMEM and ILK cDNA.The cell proliferation was detected by XTT assay and the mRNA and protein expressionsof ILK and α-SMA were detected by Real-time qPCR and Western blot.Results ① XTT results showed that the cellular proliferation level after 48 h in four groups were 0.820 ± 0.065,0.873 ± 0.041,0.554 ± 0.013 and 1.296 ± 0.094,respectively.The cellular proliferation curve showed that the cellular proliferatiion level was very flat in ILK siRNA group while the cellular proliferation level gradually increased from 12 h.48 h after transfection,the cellular proliferation level in ILK siRNA group was significant lower than those in other groups(P value were 0.021,0.034,0),while the cellular proliferation level in ILK cDNA group was the highest among all 4 groups(P value were 0.017,0.009,0).②The Real-time qPCR showed that the expressions of ILK mRNA and α-SMA mRNA were 0.693 ± 0.412 and 0.422 ± 0.037 in control group,were 0.621 ±0.183 and 0.388 ±0.005 in jetPRIMETM group,were 0.052 ±0.019 and 0.073 ±0.023 in ILK siRNA group,were 240.193 ±35.170 and 138.056 ±24.060 in ILK cDNA group.The expressions of ILK mRNA and α-SMA mRNA in ILK siRNA group were significantly lower than those in other three groups(P ＜ 0.05).And the expressions of ILK mRNA and α-SMA mRNA in ILK cDNA group were significantly higher than those in other three groups(P ＜ 0.05).③The Western blot also showed that the expression of ILK and α-SMA proteins were decreased in ILK siRNA group and increased in ILK cDNA group.Conclusion ILK may promote the proliferation and differentiation of human scar fibroblast.It may play an important role in scar formation and contracture.
