中华烧伤杂志
中華燒傷雜誌
중화소상잡지
16
2011年
6期
422-426
,共5页
谢有富%张俊成%刘思隽%戴丽冰%杜高伟
謝有富%張俊成%劉思雋%戴麗冰%杜高偉
사유부%장준성%류사준%대려빙%두고위
褪黑激素%成纤维细胞%细胞增殖%细胞凋亡
褪黑激素%成纖維細胞%細胞增殖%細胞凋亡
퇴흑격소%성섬유세포%세포증식%세포조망
Melatonin%Fibroblasts%Cell proliferation%Apoptosis%Hypertrophic scar
目的 探讨褪黑激素对人增生性瘢痕Fb增殖和凋亡的影响与机制. 方法 采用组织块法分离培养人增生性瘢痕Fb.按随机数字表法将细胞分为低、中、高浓度组和对照组.前3组细胞分别采用含1 × 10-5、1×10-3、1 mmol/L褪黑激素的培养液培养,对照组不加褪黑激素常规培养.处理后24 h进行如下检测:对各组细胞进行形态学观察；用四氮唑复合物( XTT)-硫酸酚嗪甲酯(PMS)比色法检测细胞增殖活性；对细胞行膜联蛋白V-异硫氰酸荧光素(FITC)和碘化丙啶(PI)双染色后,用流式细胞仪分析细胞周期和凋亡情况；荧光定量RT-PCR法检测细胞周期蛋白E(cyclin E)、p53和Fas mRNA表达量.对数据行方差分析和LSD检验. 结果 形态学观察显示,对照组Fb为长梭形,呈集落分布；3个浓度组Fb随着褪黑激素浓度升高,细胞逐渐分散,胞体变形缩小,胞膜皱缩,核质比例减小.对照组、低浓度组、中浓度组、高浓度组Fb增殖活性(吸光度值)依次下降,分别为1.79±0.10、1.49±0.15、1.24±0.20、0.92±0.09(F=67.61,P ＜0.05)；S期细胞百分比依次下降,分别为(16.9±1.3)％、(10.6±1.1)％、(6.1±1.2)％、(3.2±0.8)％(F=286.10,P ＜0.05);G2/M期细胞百分比依次下降,分别为(16.7±1.6)％、(13.5±1.1)％、(9.8±1.0)％(6.0±0.7)％(F=162.69,P＜0.05);早、晚期凋亡细胞百分比依次升高(F值分别为424.05、236.44,P值均小于0.05).对照组、低浓度组、中浓度组、高浓度组细胞cyclin E的mRNA表达量依次下降,分别为2.90±0.30、1.58±0.21、0.90±0.20、0.24±0.12(F=266.79,P＜0.05)；p53和Fas mRNA表达量依次升高(F值分别为10.11、12.03,P值均小于0.05). 结论 褪黑激素可通过影响细胞cyclin E、p53和Fas基因的表达,抑制增生性瘢痕Fb增殖并诱导该细胞凋亡.
目的 探討褪黑激素對人增生性瘢痕Fb增殖和凋亡的影響與機製. 方法 採用組織塊法分離培養人增生性瘢痕Fb.按隨機數字錶法將細胞分為低、中、高濃度組和對照組.前3組細胞分彆採用含1 × 10-5、1×10-3、1 mmol/L褪黑激素的培養液培養,對照組不加褪黑激素常規培養.處理後24 h進行如下檢測:對各組細胞進行形態學觀察；用四氮唑複閤物( XTT)-硫痠酚嗪甲酯(PMS)比色法檢測細胞增殖活性；對細胞行膜聯蛋白V-異硫氰痠熒光素(FITC)和碘化丙啶(PI)雙染色後,用流式細胞儀分析細胞週期和凋亡情況；熒光定量RT-PCR法檢測細胞週期蛋白E(cyclin E)、p53和Fas mRNA錶達量.對數據行方差分析和LSD檢驗. 結果 形態學觀察顯示,對照組Fb為長梭形,呈集落分佈；3箇濃度組Fb隨著褪黑激素濃度升高,細胞逐漸分散,胞體變形縮小,胞膜皺縮,覈質比例減小.對照組、低濃度組、中濃度組、高濃度組Fb增殖活性(吸光度值)依次下降,分彆為1.79±0.10、1.49±0.15、1.24±0.20、0.92±0.09(F=67.61,P ＜0.05)；S期細胞百分比依次下降,分彆為(16.9±1.3)％、(10.6±1.1)％、(6.1±1.2)％、(3.2±0.8)％(F=286.10,P ＜0.05);G2/M期細胞百分比依次下降,分彆為(16.7±1.6)％、(13.5±1.1)％、(9.8±1.0)％(6.0±0.7)％(F=162.69,P＜0.05);早、晚期凋亡細胞百分比依次升高(F值分彆為424.05、236.44,P值均小于0.05).對照組、低濃度組、中濃度組、高濃度組細胞cyclin E的mRNA錶達量依次下降,分彆為2.90±0.30、1.58±0.21、0.90±0.20、0.24±0.12(F=266.79,P＜0.05)；p53和Fas mRNA錶達量依次升高(F值分彆為10.11、12.03,P值均小于0.05). 結論 褪黑激素可通過影響細胞cyclin E、p53和Fas基因的錶達,抑製增生性瘢痕Fb增殖併誘導該細胞凋亡.
목적 탐토퇴흑격소대인증생성반흔Fb증식화조망적영향여궤제. 방법 채용조직괴법분리배양인증생성반흔Fb.안수궤수자표법장세포분위저、중、고농도조화대조조.전3조세포분별채용함1 × 10-5、1×10-3、1 mmol/L퇴흑격소적배양액배양,대조조불가퇴흑격소상규배양.처리후24 h진행여하검측:대각조세포진행형태학관찰；용사담서복합물( XTT)-류산분진갑지(PMS)비색법검측세포증식활성；대세포행막련단백V-이류청산형광소(FITC)화전화병정(PI)쌍염색후,용류식세포의분석세포주기화조망정황；형광정량RT-PCR법검측세포주기단백E(cyclin E)、p53화Fas mRNA표체량.대수거행방차분석화LSD검험. 결과 형태학관찰현시,대조조Fb위장사형,정집락분포；3개농도조Fb수착퇴흑격소농도승고,세포축점분산,포체변형축소,포막추축,핵질비례감소.대조조、저농도조、중농도조、고농도조Fb증식활성(흡광도치)의차하강,분별위1.79±0.10、1.49±0.15、1.24±0.20、0.92±0.09(F=67.61,P ＜0.05)；S기세포백분비의차하강,분별위(16.9±1.3)％、(10.6±1.1)％、(6.1±1.2)％、(3.2±0.8)％(F=286.10,P ＜0.05);G2/M기세포백분비의차하강,분별위(16.7±1.6)％、(13.5±1.1)％、(9.8±1.0)％(6.0±0.7)％(F=162.69,P＜0.05);조、만기조망세포백분비의차승고(F치분별위424.05、236.44,P치균소우0.05).대조조、저농도조、중농도조、고농도조세포cyclin E적mRNA표체량의차하강,분별위2.90±0.30、1.58±0.21、0.90±0.20、0.24±0.12(F=266.79,P＜0.05)；p53화Fas mRNA표체량의차승고(F치분별위10.11、12.03,P치균소우0.05). 결론 퇴흑격소가통과영향세포cyclin E、p53화Fas기인적표체,억제증생성반흔Fb증식병유도해세포조망.
Objective To study the effect of melatonin on proliferation and apoptosis of fibrohlasts in human hypertrophic scar and its mechanism.Methods Fibroblasts from human hypertrophic scar were isolated and cultured with DMEM medium containing 10％ FBS,and then they were divided into control (C,added with ethanol),low concentration (LC,added with 1 × 10 -5 mmol/L melatonin),middle concentration (MC,added with 1 × 10-3 mmol/L melatonin),and high concentration (HC,added with 1 mmol/L melatonin) groups according to the random number table.After being cultured for 24 hours,cell morphologic change was observed under microscope; XTT-PMS assay was used to examine cell proliferative activity; cell cycle and apoptosis were assessed with flow eytometry after double staining of FITC and PI,and the levels of cyclin E,p53,and Fas mRNA were determined with fluorescence quantitative RT-PCR.Data were processed with analysis of variance and LSD test.Results ( 1 ) Fibroblasts in C group were spindie-shaped with growth in colonies.Along with the increase in melatonin concentration,fibroblasts in LC,MC,and HC groups gradually dispesed,deformed and atrophied,with shrunk cellular membrane,and decrease in ratio of nucleus and cytoplasm.(2) Proliferative activity of fibroblasts in LC,MC,and HC groups decreased along with an increase in melatonin concentration ( 1.49 ± 0.15,1.24 ± 0.20,and 0.92 ± 0.09 ),which were lower that in C group ( 1.79 ± 0.10,F =67.61,P ＜0.05).Cell ratios of S and G2/M phases in LC,MC,and HC groups decreased along with an increase in melatonin concentration,which were all lower than those in C group [(10.6±1.1)％,(6.1 ±1.2)％,(3.2±0.8)％ vs.(16.9±1.3)％,F=286.10,P ＜0.05; (13.5±1.1)％,(9.8±1.0)％,(6.0±0.7)％ vs.(16.7±1.6)％.F =162.69,P ＜0.05].Apoptotic rates in early and late stages of LC,MC,and HC groups increased along with an increase in melatonin concentration,all higher than those in C group (with F value respectively 424.05,236.44,P values all below 0.05 ).The expressions of cyclin E mRNA in LC,MC,and HC groups decreased along with an increase in melatonin concentration,which were lower than that in C group (1.58 ± 0.21,0.90±0.20,and0.24±0.12 vs.2.90±0.30,F =266.79,P ＜0.05),while the expressions of p53 mRNA and Fas mRNA showed opposite tendency (with F value respectively 10.11,12.03,P values all below 0.05). Conclusions Melatonin can inhibit proliferation and induce apoptosis of fibroblasts in hypertrophic scar through regulating the gene expressions of cyclin E,p53,and Fas.
