丝裂霉素对体外培养人正常皮肤成纤维细胞及Hacat细胞的作用及机制研究
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摘要
目的:探讨不同浓度的丝裂霉素对体外培养的人正常皮肤成纤维细胞及HaCat细胞形态、细胞增殖、生长特性及细胞凋亡等方面的影响,从而选取适当的作用浓度,研究该浓度的丝裂霉素作用于皮肤成纤维细胞后,细胞内caspase-3和caspase-8的表达,初步探讨丝裂霉素的凋亡诱导机制,以及成纤维细胞内细胞因子TGF-β1、bFGF与Ⅰ、Ⅲ型前胶原蛋白mRNA的表达变化,为局部应用丝裂霉素预防和治疗耳鼻咽喉术后皮肤纤维增生及瘢痕形成提供理论依据。
方法:体外培养人正常皮肤成纤维细胞及HaCat细胞,以0.04mg/ml、0.4mg/ml浓度的丝裂霉素及无血清培养基对照分别作用于这两种细胞5min,培养不同时程后采用倒置显微镜、细胞计数及MTT检测法观察两种细胞形态、增殖、生长特性与增殖抑制情况;采用AO/PI染色观察计数成纤维细胞的凋亡率;通过western blot法检测caspase-3的表达,免疫荧光法检测细胞内caspase-8活化及TGF-β1的表达;RT-PCR法检测细胞内TGF-β1、bFGF、Ⅰ型及Ⅲ型前胶原蛋白mRNA的表达情况。采用IPP图像分析软件和spssv15.0统计软件进行图片分析与统计学处理。
结果:正常生长的成纤维细胞及HaCat细胞呈指数生长;0.04mg/ml及0.4mg/ml浓度的丝裂霉素作用5min后能有效抑制皮肤成纤维细胞及HaCat细胞的增殖,并使细胞增大变形、细胞间形态差异较大。0.04mg/ml浓度的丝裂霉素作用后第5天成纤维细胞呈现小幅增殖,对成纤维细胞的抑制强度明显低于0.4mg/ml浓度的丝裂霉素;两种试验浓度的丝裂霉素所致成纤维细胞的死亡形式主要为凋亡,0.4mg/ml浓度的丝裂霉素促凋亡作用明显强于0.04mg/ml浓度的丝裂霉素,两者比较差异有统计学意义(p<0.05)。0.4mg/ml浓度的丝裂霉素作用后前caspase-3表达先升高后降低,免疫荧光法检测到随培养时间的延长caspase-8活化片段的荧光强度逐渐增强,提示caspase-8的活化增多;0.4mg/ml浓度的丝裂霉素作用后,免疫荧光法检测细胞内TGF-β1蛋白表达变化不明显,但细胞内TGF-β1 mRNA却随培养时间的延长表达逐渐减少,bFGF mRNA的表达逐渐增高,Ⅰ型、Ⅲ型前胶原蛋白mRNA的表达量则明显下降。
结论:0.04mg/ml和0.4mg/ml浓度的丝裂霉素能有效的抑制人皮肤成纤维细胞增殖,以0.4mg/ml浓度的丝裂霉素抑制作用较持久;丝裂霉素可以通过启动caspase凋亡途径,增加细胞凋亡;实验浓度的丝裂霉素可以从mRNA水平调节细胞因子TGF-β1、bFGF以及Ⅰ型、Ⅲ型前胶原蛋白的表达,从而减少细胞外机基质的合成,有效的抑制纤维增生瘢痕形成。但与此同时,丝裂霉素对HaCat细胞也有较强的增殖抑制作用,提示临床局部应用丝裂霉素预防和治疗耳鼻咽喉术后皮肤纤维增生及瘢痕形成时应注意保护表皮细胞,避免其毒副作用的影响。
Objective:To investigate the effects of mitomycin on cultured human normal skin fibroblast and HaCat cell,including cellular morphology,cell proliferation,growth characteristics and apoptosis,in order to find out the clinically relevant concentrations of mitomycin.With the concentration,we try to detect the expression changes of intracellular caspase-3,capase-8,TGF-β1 on protein level and TGF-β1,bFGF, procollagenⅠand procollagenⅢon mRNA level separately,which can give theoretical supports to the clinical application of mitomycin.
Methods:Cultured human normal skin fibroblast and HaCat cell in vitro,mitomycin was applied to the cells for 5 minutes with concertrations of 0.04mg/ml or 0.4mg/ml,and cultural media without serum was used as control,then the cellular morphology change,growth characteristics,cell proliferation were observed by inverted microscope,AO staining,cell count and MTT test at different intervals.The apoptosis rate of fibroblast was detected with AO/PI staining.For fibroblast,western blot was used to detect caspase-3 expression.Immunofluorescense was used to detect caspase-8 activation and intracellular TGF-β1 expression.RT-PCR was used to detect the mRNA expression of TGF-β1,bFGF,procollagenⅠand procollagenⅢ.IPP was used to analyze picture and SPSS 15.0 was used to make statistical analysis.
Result:The cultured normal human skin fibroblast and HaCat cell grew with a exponential phase.Mitomycin could inhibit the proliferation of both fibroblast and HaCat cell dose-dependently,and it could make the cells grow bigger and irregular.5 days after the 5min exposure of mitomycin at 0.04mg/ml,fibroblast got a tiny proliferation,the inhibition effect of mitomycin at 0.04mg/ml was weaker than 0.4mg/ml(p＜0.05). Meanwhile,application of mitomycin at either 0.04mg/ml or 0.4mg/ml for 5min induced fibroblasts apoptosis but not necrosis.the higher concentration of mytomycin is,the higher apoptosis rate of fibroblast is (p＜0.05).After 0.4mg/ml mitomycin's application,the quantity of intracellular pre-caspase-3 rose at first,and then fell down. Immunofluorescense test showed that:the immunofluorescense intensity of active caspase-8 increased gradually after mitomycin's application,and there is no significant change in TGF-β1's immunofluorescense intensity. But the mRNA level of TGF-β1 decreased obviously after mitomycin's application,as well as procollagenⅠand procollagenⅢ.In contrary, bFGF increased on mRNA level.
Conclusion:Mitomycin could inhibit fibroblast proliferation and increase its apoptosis through activating caspase pathway.Mitomycin could regulate the expression of TGF-β1 and bFGF,and decrease procollagenⅠandⅢexpression on mRNA level,which might reduce extracellular matrix synthesis and lessen scar formation.But mitomycin had even stronger inhibition effect on HaCat cell,so we sould pay more attention to protect epithelial cell to avoid mitomycin's side effect.
方法:体外培养人正常皮肤成纤维细胞及HaCat细胞,以0.04mg/ml、0.4mg/ml浓度的丝裂霉素及无血清培养基对照分别作用于这两种细胞5min,培养不同时程后采用倒置显微镜、细胞计数及MTT检测法观察两种细胞形态、增殖、生长特性与增殖抑制情况;采用AO/PI染色观察计数成纤维细胞的凋亡率;通过western blot法检测caspase-3的表达,免疫荧光法检测细胞内caspase-8活化及TGF-β1的表达;RT-PCR法检测细胞内TGF-β1、bFGF、Ⅰ型及Ⅲ型前胶原蛋白mRNA的表达情况。采用IPP图像分析软件和spssv15.0统计软件进行图片分析与统计学处理。
结果:正常生长的成纤维细胞及HaCat细胞呈指数生长;0.04mg/ml及0.4mg/ml浓度的丝裂霉素作用5min后能有效抑制皮肤成纤维细胞及HaCat细胞的增殖,并使细胞增大变形、细胞间形态差异较大。0.04mg/ml浓度的丝裂霉素作用后第5天成纤维细胞呈现小幅增殖,对成纤维细胞的抑制强度明显低于0.4mg/ml浓度的丝裂霉素;两种试验浓度的丝裂霉素所致成纤维细胞的死亡形式主要为凋亡,0.4mg/ml浓度的丝裂霉素促凋亡作用明显强于0.04mg/ml浓度的丝裂霉素,两者比较差异有统计学意义(p<0.05)。0.4mg/ml浓度的丝裂霉素作用后前caspase-3表达先升高后降低,免疫荧光法检测到随培养时间的延长caspase-8活化片段的荧光强度逐渐增强,提示caspase-8的活化增多;0.4mg/ml浓度的丝裂霉素作用后,免疫荧光法检测细胞内TGF-β1蛋白表达变化不明显,但细胞内TGF-β1 mRNA却随培养时间的延长表达逐渐减少,bFGF mRNA的表达逐渐增高,Ⅰ型、Ⅲ型前胶原蛋白mRNA的表达量则明显下降。
结论:0.04mg/ml和0.4mg/ml浓度的丝裂霉素能有效的抑制人皮肤成纤维细胞增殖,以0.4mg/ml浓度的丝裂霉素抑制作用较持久;丝裂霉素可以通过启动caspase凋亡途径,增加细胞凋亡;实验浓度的丝裂霉素可以从mRNA水平调节细胞因子TGF-β1、bFGF以及Ⅰ型、Ⅲ型前胶原蛋白的表达,从而减少细胞外机基质的合成,有效的抑制纤维增生瘢痕形成。但与此同时,丝裂霉素对HaCat细胞也有较强的增殖抑制作用,提示临床局部应用丝裂霉素预防和治疗耳鼻咽喉术后皮肤纤维增生及瘢痕形成时应注意保护表皮细胞,避免其毒副作用的影响。
Objective:To investigate the effects of mitomycin on cultured human normal skin fibroblast and HaCat cell,including cellular morphology,cell proliferation,growth characteristics and apoptosis,in order to find out the clinically relevant concentrations of mitomycin.With the concentration,we try to detect the expression changes of intracellular caspase-3,capase-8,TGF-β1 on protein level and TGF-β1,bFGF, procollagenⅠand procollagenⅢon mRNA level separately,which can give theoretical supports to the clinical application of mitomycin.
Methods:Cultured human normal skin fibroblast and HaCat cell in vitro,mitomycin was applied to the cells for 5 minutes with concertrations of 0.04mg/ml or 0.4mg/ml,and cultural media without serum was used as control,then the cellular morphology change,growth characteristics,cell proliferation were observed by inverted microscope,AO staining,cell count and MTT test at different intervals.The apoptosis rate of fibroblast was detected with AO/PI staining.For fibroblast,western blot was used to detect caspase-3 expression.Immunofluorescense was used to detect caspase-8 activation and intracellular TGF-β1 expression.RT-PCR was used to detect the mRNA expression of TGF-β1,bFGF,procollagenⅠand procollagenⅢ.IPP was used to analyze picture and SPSS 15.0 was used to make statistical analysis.
Result:The cultured normal human skin fibroblast and HaCat cell grew with a exponential phase.Mitomycin could inhibit the proliferation of both fibroblast and HaCat cell dose-dependently,and it could make the cells grow bigger and irregular.5 days after the 5min exposure of mitomycin at 0.04mg/ml,fibroblast got a tiny proliferation,the inhibition effect of mitomycin at 0.04mg/ml was weaker than 0.4mg/ml(p＜0.05). Meanwhile,application of mitomycin at either 0.04mg/ml or 0.4mg/ml for 5min induced fibroblasts apoptosis but not necrosis.the higher concentration of mytomycin is,the higher apoptosis rate of fibroblast is (p＜0.05).After 0.4mg/ml mitomycin's application,the quantity of intracellular pre-caspase-3 rose at first,and then fell down. Immunofluorescense test showed that:the immunofluorescense intensity of active caspase-8 increased gradually after mitomycin's application,and there is no significant change in TGF-β1's immunofluorescense intensity. But the mRNA level of TGF-β1 decreased obviously after mitomycin's application,as well as procollagenⅠand procollagenⅢ.In contrary, bFGF increased on mRNA level.
Conclusion:Mitomycin could inhibit fibroblast proliferation and increase its apoptosis through activating caspase pathway.Mitomycin could regulate the expression of TGF-β1 and bFGF,and decrease procollagenⅠandⅢexpression on mRNA level,which might reduce extracellular matrix synthesis and lessen scar formation.But mitomycin had even stronger inhibition effect on HaCat cell,so we sould pay more attention to protect epithelial cell to avoid mitomycin's side effect.
引文
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