EGFR抑制剂对UVA照射皮肤成纤维细胞保护作用的实验研究
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摘要
【研究背景及目的】
随着人们寿命的延长和接受外界刺激的增加,皮肤老化的研究成为了一个研究热点,人们希望有健康的皮肤和看起来更年轻的皮肤。皮肤老化分为内源性老化和外源性老化,特征性改变即皱纹,色素沉着和皮肤纹理的消失。内源性因素引起的皮肤老化是不可避免的自然过程,是随着时间的程序性变化,也称为自然老化。外源性因素主要是环境因素,如：紫外线,应激和环境污染。其中紫外线的作用是最主要和最突出的,由于皮肤反复暴露于紫外线中造成的皮肤老化也称为光老化(Photoaging)~([1])。皮肤光老化最特征性的表现是在面颈部皱纹的形成,这是由于胶原的快速降解导致皮肤弹性下降引起的~([2])。
紫外线中的长波紫外线(UVA)与人体皮肤光老化发生关系密切,在皮肤光老化的研究日显重要。紫外线照射等各种因素通过配体独立的信号机制激活了细胞各种受体信号转导路径。成纤维细胞是真皮中最主要的细胞成分,紫外线(UV)照射导致成纤维细胞的过早衰老,胶原合成能力下降,基质金属蛋白酶(MMPs)表达升高,细胞外基质降解,胶原降解和合成的平衡被打破,形成了光老化的皱纹特征~([3])。在皮肤光老化发生的机制方面,主要从胶原的合成和降解方面进行大量研究。近年来,国内外学者针对UVA照射导致细胞内的一系列变化进行了大量研究,AP-1蛋白表达在光老化的研究中有着重要作用,其由c-fos和c-jun蛋白组成~([4])。国内外进行了大量抗光老化的研究,但至今尚未找到确切的抗光老化方法。随着对光老化发生机制的深入研究,研究者发现细胞因子之间的调控对皮肤光老化起着很重要的作用,细胞膜上细胞因子受体参与光老化的进程,细胞因子及细胞因子受体通路在光老化中的作用越来越受到重视。PallaviChauhan研究表明,各种刺激条件,如：UV照射,渗透性应激和热休克等会导致表皮生长因子受体,白细胞因子受体,肿瘤坏死因子受体,血小板源性生长因子和活化因子受体的信号转导通路激活。它们激活后导致基质金属蛋白酶分泌增加,胶原降解,最终导致皮肤皱纹的形成。在五个信号通路的发生过程中,AP-1转录因子中的c-jun蛋白表达在通路中的常见调节蛋白~([5])。信号路径中AP-1蛋白的表达在光老化发生过程中发挥重要作用~([6])。如何应用细胞因子等物质进行抗光老化研究,最近的研究对这方面的报道甚少。
本研究旨在进行体外人皮肤成纤维细胞培养,构建细胞光老化模型,探讨不同剂量UVA照射体外培养的皮肤成纤维细胞,对其形态和增殖情况的影响及对细胞内AP-1蛋白的组成部分(c-fos和c-jun)表达的影响。同时用不同剂量的EGFR抑制剂预处理成纤维细胞后,进行UVA 15 J/cm2照射,通过表皮生长因子受体通路的干预,观察细胞形态和检测细胞增殖情况,检测细胞AP-1蛋白的组成成分c-fos和c-jun表达的变化。通过以上研究,全面系统地探讨EGFR抑制剂对UVA照射体外培养皮肤成纤维细胞光老化的保护作用。
【研究方法】
1.取年轻成人包皮,用组织块培养法进行皮肤成纤维细胞培养传代,用第5代成纤维细胞做实验对象,进行细胞分组。采用不同剂量的UVA(5 J/cm210J/cm~2、20J/cm~2)照射,设立对照组(未照射),建立成纤维细胞光老化模型。培养24h后采用四甲基偶氮唑盐比色(MTT)法检测照射后细胞的增殖活性,光学倒置显微镜观察细胞形态。采用细胞免疫组化方法检测不同剂量UVA照射对体外培养的皮肤成纤维细胞c-fos,c-jun表达的影响。
2.分别加入不同剂量EGFR抑制剂于培养的成纤维细胞中,即小剂量(0.1ug/ml)、中剂量(1.0 ug/m1)、大剂量(10.0 ug/ml),再用15J/cm~2UVA照射,MTT法检测照射后细胞增殖活性,光学显微镜观察细胞形态。分别采用细胞免疫组化方法及蛋白质印迹技术检测EGFR抑制剂对UVA照射体外培养的皮肤成纤维细胞c-fos,c-jun蛋白表达的影响。
【研究结果】
1.不同剂量的UVA(小剂量5 J/cm~2、中剂量10J/cm~2、大剂量20J/cm~2)照射体外培养的皮肤成纤维细胞结果显示：随着UVA照射剂量增加,成纤维细胞OD值下降。与对照组比较,UVA照射大、中剂量组OD值明显下降,差异有统计学意义(P<0.01)。不同剂量UVA照射后,细胞形态变圆,细胞间隙加大,失去原来的梭形,偶见细胞变圆悬浮于液体中。细胞免疫组化检测显示：随着UVA照射剂量增加,c-fos.c-jun蛋白细胞免疫组化染色均为阳性,染色为棕黄色。在细胞胞浆和细胞核,OD值随着照射剂量增加而上升。与对照组比较,UVA照射大中剂量组c-fos和c-jun蛋白表达差异有统计学意义(P<0.05),小剂量组与对照组比较,差异无统计学意义(P>0.05)。
2.本实验通过UVA15 J/cm2照射加入EGFR抑制剂不同剂量的成纤维细胞,即小剂量(0.01ug/ml)、中剂量(0.1 ug/m1)、大剂量(1.0 ug/m])组预处理,MTT结果显示：随着EGFR抑制剂的剂量增加,各剂量组的OD值高于对照组,但差异无统计学意义(P>0.05),对于细胞形态也没有明显影响。细胞免疫组化显示：c-fos细胞免疫组化染色均为阳性,在胞浆和胞核,OD值随照射计量增加而降低,大,中剂量组的OD值明显高于对照组,差异有统计学意义(P<0.05)。c-jun蛋白表达与对照组比较,差异无统计学意义。而蛋白质印迹技术实验结果表明：蛋白质印迹技术检测到细胞内c-fos,c-jun蛋白,大剂量组和中剂量组与照射组比较,差异均有统计学意义(P<0.05)。小剂量组与照射组比较,差异无统计学意义(P>0.05)。
【结论】
1.UVA照射体外培养的皮肤成纤维细胞,导致细胞衰老,增殖活性下降,细胞由原来的梭形变为圆形,同时激活细胞内AP-1蛋白(c-fos.c-jun)高表达,作为光老化关键信号路径中的的AP-1蛋白(c-fos.c-jun)的高表达,将会引起下游信号的基质金属蛋白酶的表达增高,对胶原降解增加,加剧光老化的发生。
2.在UVA照射前加入EGFR抑制剂,对于体外培养皮肤成纤维细胞增殖活性和细胞形态没有明显影响。通过干预细胞内信号转导，使得细胞内AP-1蛋白(c-fos.c-jun)的表达降低。与单纯照射组比较,干预后细胞内c-fos,c-jun蛋白表达降低,使得成纤维细胞的胶原合成和降解保持平衡状态,从而对成纤维细胞光老化起防护作用,达到抗光老化的目的。
【Background and Objective】
Skin ageing is one of the most demanding areas of research, and now it is more significant to prolong health expectancy, allowing people to look younger and live healthy lives rather than to prolong life expectancy. Skin ageing is characterized by signs like discoloration, wrinkles and texture loss. The two major types of skin ageing are intrinsic ageing and extrinsic ageing. Intrinsic ageing is genetically programmed ageing, also called chronological ageing, whereas extrinsic ageing is due to environmental factors like sunlight, mainly UV rays (commonly known as photoageing), stress and pollution. The skin aging caused by exposure to sunlight is named photoaging. It is characterized with coarse, skin laxitas, deep and severe wrinkling, freckles, telangiectasia and pigmentary changes or even cancer on exposed areas such as the face, neck and forearm.
Fibroblasts are the most important components of cells in skin dermal. Ultraviolet (UV) irradiation leads fibroblasts to earlier aging. It decreases the synthesis of collagen and increases the expression of matrix metalloproteinases.These lead to decrease of extracellular matrix. In a word, it leads to skin photoaging. The ultraviolet A (UVA) in ultraviolet light is closely related to human skin photoaging and has attracted worldwide attention. As to skin photoaging mechanisms, the research about synthesis and degradation of collagen are more important.
UVA irradiation causes a series of changes within the cell.The AP-1 protein expression in the photoaging research plays an important role. The most important composition of AP-1 protein are c-fos and c-jun.With development of studying on the anti-aging mechanism, researchers have found that the cytokine and the cytokine receptor plays a very important role in regulating skin-aging, and have devoted more attention to it's study. However, the study about using cytokines to anti-skin aging are inadequate.
In this study, we use fibroblast and UVA to establish photo-aging model.We use different dose of UVA iridiate fibroblast. Then we test cell proliferation activity by MTT. The expression of c-fos and c-jun detected by immunocytochemistry and Western Blot method. At the same time with different doses of EGFR inhibitors treat fibroblasts and use UVA 15 J/cm2 irradiate fibroblastd.Then we observe cell morphology and cell proliferation, detect the changes of c-fos and c-jun expression. We discuss whether or not EGFR Inhibitor inhibits the expression of c-fos and c-jun and protect fibroblasts from UVA.
【Methods】
1. We culture fibroblasts of foreskin from young adults and using the five generation cells for experiment. We divide cells into four groups and use different doses of UVA (0 J/cm~2,5 J/cm~2,10J/cm~2,20J/cm~2) irradiate cells. Then we test cell proliferation activity by MTT, and use immunocytochemistry method to test expression of c-fos and c-jun protein in fibroblast.
2. We use the five generetion fibroblasts to do experiment and divided to five groups. By adding different doses of EGFR Inhibitor, small dose (0.1 ug/ml), medium dose (1.0 ug/ml), high dose (10.0 ug/ml) to the cultured fibroblasts, and use UVA15J/cm~2 irradiation.Then we test cell proliferation activity.by MTT, use immunocytochemistry and Western blot method to test expression of c-fos and c-jun protein in fibroblasts.
【Results】
1. Different doses of UVA (the small dose 5 J/cm2, the medium dose 10J/cm~2, high-dose 20J/cm~2) irradiation skin fibroblasts in vitro showes that:with the UVA radiation dose increased, fibroblast OD values decreased. Comparing the middle and high doses group with control group, the OD values have a very significant difference (p<0.01). The small dose group have a significant difference (p<0.05). Using different doses of UVA irradiate cells, the cells shape became round, the gap between cells increased, lose the original shuttle shape. Immunocytochemical result showes that:with the UVA radiation dose increased, c-fos, c-jun were positive in the cytoplasm and nucleus, OD values increased with the dose increased.Compared with control group, UVA 10 J/cm`2, UVA 20 J/cm~2 irradiation c-fos and c-jun expression was significantly different (P<0.05),5 J/cm~2 dose group has not significantly different (P>0.05).
2.We treat the fibroblast with different doses of EGFR inhibitors, small dose(0. 1ug/ml), medium dose (1.0 ug/ml), high-dose (10.0 ug/ml) and irradiated by UVA15 J/cm2. The results of MTT show that:with the EGFR inhibitor increase, OD values of fibroblasts increased compared with irradiation group, but have not statistically significance(P>0.05). It had not significant effect on cell morphology. Immunocytochemistry showes that: c-fos staining is positive in the cytoplasm and nucleus, OD values decreased with EGFR inhibitor increased. But the c-jun expression have not statistically significant between experiment group and the control group(P>0.05). The Western blot results showed that:for c-fos and c-jun protein, compared with the control group, medium and high-dose group have statistically significant (P<0.05).
【Conclusion】
1. Cultured skin fibroblasts, UVA radiation cause fibroblast aging, the proliferation decreased, and the shape changed from the spindle to round cells, while AP-1 activation of intracellular protein (c-fos, c-jun) highly expressed.
2. We treat the fibroblast with different doses of EGFR inhibitors and irradiated by UVA15 J/cm2.This could not improve skin fibroblast proliferation activity in vitro, and no change in cells'morphology. And after treated by EGFR inhibitors, intracellular c-fos, c-jun expression decreased. This will induce the synthesis of MMPs and keep the balance of collagen.This protect the fibroblasts from the photoaging of UVA.
随着人们寿命的延长和接受外界刺激的增加,皮肤老化的研究成为了一个研究热点,人们希望有健康的皮肤和看起来更年轻的皮肤。皮肤老化分为内源性老化和外源性老化,特征性改变即皱纹,色素沉着和皮肤纹理的消失。内源性因素引起的皮肤老化是不可避免的自然过程,是随着时间的程序性变化,也称为自然老化。外源性因素主要是环境因素,如：紫外线,应激和环境污染。其中紫外线的作用是最主要和最突出的,由于皮肤反复暴露于紫外线中造成的皮肤老化也称为光老化(Photoaging)~([1])。皮肤光老化最特征性的表现是在面颈部皱纹的形成,这是由于胶原的快速降解导致皮肤弹性下降引起的~([2])。
紫外线中的长波紫外线(UVA)与人体皮肤光老化发生关系密切,在皮肤光老化的研究日显重要。紫外线照射等各种因素通过配体独立的信号机制激活了细胞各种受体信号转导路径。成纤维细胞是真皮中最主要的细胞成分,紫外线(UV)照射导致成纤维细胞的过早衰老,胶原合成能力下降,基质金属蛋白酶(MMPs)表达升高,细胞外基质降解,胶原降解和合成的平衡被打破,形成了光老化的皱纹特征~([3])。在皮肤光老化发生的机制方面,主要从胶原的合成和降解方面进行大量研究。近年来,国内外学者针对UVA照射导致细胞内的一系列变化进行了大量研究,AP-1蛋白表达在光老化的研究中有着重要作用,其由c-fos和c-jun蛋白组成~([4])。国内外进行了大量抗光老化的研究,但至今尚未找到确切的抗光老化方法。随着对光老化发生机制的深入研究,研究者发现细胞因子之间的调控对皮肤光老化起着很重要的作用,细胞膜上细胞因子受体参与光老化的进程,细胞因子及细胞因子受体通路在光老化中的作用越来越受到重视。PallaviChauhan研究表明,各种刺激条件,如：UV照射,渗透性应激和热休克等会导致表皮生长因子受体,白细胞因子受体,肿瘤坏死因子受体,血小板源性生长因子和活化因子受体的信号转导通路激活。它们激活后导致基质金属蛋白酶分泌增加,胶原降解,最终导致皮肤皱纹的形成。在五个信号通路的发生过程中,AP-1转录因子中的c-jun蛋白表达在通路中的常见调节蛋白~([5])。信号路径中AP-1蛋白的表达在光老化发生过程中发挥重要作用~([6])。如何应用细胞因子等物质进行抗光老化研究,最近的研究对这方面的报道甚少。
本研究旨在进行体外人皮肤成纤维细胞培养,构建细胞光老化模型,探讨不同剂量UVA照射体外培养的皮肤成纤维细胞,对其形态和增殖情况的影响及对细胞内AP-1蛋白的组成部分(c-fos和c-jun)表达的影响。同时用不同剂量的EGFR抑制剂预处理成纤维细胞后,进行UVA 15 J/cm2照射,通过表皮生长因子受体通路的干预,观察细胞形态和检测细胞增殖情况,检测细胞AP-1蛋白的组成成分c-fos和c-jun表达的变化。通过以上研究,全面系统地探讨EGFR抑制剂对UVA照射体外培养皮肤成纤维细胞光老化的保护作用。
【研究方法】
1.取年轻成人包皮,用组织块培养法进行皮肤成纤维细胞培养传代,用第5代成纤维细胞做实验对象,进行细胞分组。采用不同剂量的UVA(5 J/cm210J/cm~2、20J/cm~2)照射,设立对照组(未照射),建立成纤维细胞光老化模型。培养24h后采用四甲基偶氮唑盐比色(MTT)法检测照射后细胞的增殖活性,光学倒置显微镜观察细胞形态。采用细胞免疫组化方法检测不同剂量UVA照射对体外培养的皮肤成纤维细胞c-fos,c-jun表达的影响。
2.分别加入不同剂量EGFR抑制剂于培养的成纤维细胞中,即小剂量(0.1ug/ml)、中剂量(1.0 ug/m1)、大剂量(10.0 ug/ml),再用15J/cm~2UVA照射,MTT法检测照射后细胞增殖活性,光学显微镜观察细胞形态。分别采用细胞免疫组化方法及蛋白质印迹技术检测EGFR抑制剂对UVA照射体外培养的皮肤成纤维细胞c-fos,c-jun蛋白表达的影响。
【研究结果】
1.不同剂量的UVA(小剂量5 J/cm~2、中剂量10J/cm~2、大剂量20J/cm~2)照射体外培养的皮肤成纤维细胞结果显示：随着UVA照射剂量增加,成纤维细胞OD值下降。与对照组比较,UVA照射大、中剂量组OD值明显下降,差异有统计学意义(P<0.01)。不同剂量UVA照射后,细胞形态变圆,细胞间隙加大,失去原来的梭形,偶见细胞变圆悬浮于液体中。细胞免疫组化检测显示：随着UVA照射剂量增加,c-fos.c-jun蛋白细胞免疫组化染色均为阳性,染色为棕黄色。在细胞胞浆和细胞核,OD值随着照射剂量增加而上升。与对照组比较,UVA照射大中剂量组c-fos和c-jun蛋白表达差异有统计学意义(P<0.05),小剂量组与对照组比较,差异无统计学意义(P>0.05)。
2.本实验通过UVA15 J/cm2照射加入EGFR抑制剂不同剂量的成纤维细胞,即小剂量(0.01ug/ml)、中剂量(0.1 ug/m1)、大剂量(1.0 ug/m])组预处理,MTT结果显示：随着EGFR抑制剂的剂量增加,各剂量组的OD值高于对照组,但差异无统计学意义(P>0.05),对于细胞形态也没有明显影响。细胞免疫组化显示：c-fos细胞免疫组化染色均为阳性,在胞浆和胞核,OD值随照射计量增加而降低,大,中剂量组的OD值明显高于对照组,差异有统计学意义(P<0.05)。c-jun蛋白表达与对照组比较,差异无统计学意义。而蛋白质印迹技术实验结果表明：蛋白质印迹技术检测到细胞内c-fos,c-jun蛋白,大剂量组和中剂量组与照射组比较,差异均有统计学意义(P<0.05)。小剂量组与照射组比较,差异无统计学意义(P>0.05)。
【结论】
1.UVA照射体外培养的皮肤成纤维细胞,导致细胞衰老,增殖活性下降,细胞由原来的梭形变为圆形,同时激活细胞内AP-1蛋白(c-fos.c-jun)高表达,作为光老化关键信号路径中的的AP-1蛋白(c-fos.c-jun)的高表达,将会引起下游信号的基质金属蛋白酶的表达增高,对胶原降解增加,加剧光老化的发生。
2.在UVA照射前加入EGFR抑制剂,对于体外培养皮肤成纤维细胞增殖活性和细胞形态没有明显影响。通过干预细胞内信号转导，使得细胞内AP-1蛋白(c-fos.c-jun)的表达降低。与单纯照射组比较,干预后细胞内c-fos,c-jun蛋白表达降低,使得成纤维细胞的胶原合成和降解保持平衡状态,从而对成纤维细胞光老化起防护作用,达到抗光老化的目的。
【Background and Objective】
Skin ageing is one of the most demanding areas of research, and now it is more significant to prolong health expectancy, allowing people to look younger and live healthy lives rather than to prolong life expectancy. Skin ageing is characterized by signs like discoloration, wrinkles and texture loss. The two major types of skin ageing are intrinsic ageing and extrinsic ageing. Intrinsic ageing is genetically programmed ageing, also called chronological ageing, whereas extrinsic ageing is due to environmental factors like sunlight, mainly UV rays (commonly known as photoageing), stress and pollution. The skin aging caused by exposure to sunlight is named photoaging. It is characterized with coarse, skin laxitas, deep and severe wrinkling, freckles, telangiectasia and pigmentary changes or even cancer on exposed areas such as the face, neck and forearm.
Fibroblasts are the most important components of cells in skin dermal. Ultraviolet (UV) irradiation leads fibroblasts to earlier aging. It decreases the synthesis of collagen and increases the expression of matrix metalloproteinases.These lead to decrease of extracellular matrix. In a word, it leads to skin photoaging. The ultraviolet A (UVA) in ultraviolet light is closely related to human skin photoaging and has attracted worldwide attention. As to skin photoaging mechanisms, the research about synthesis and degradation of collagen are more important.
UVA irradiation causes a series of changes within the cell.The AP-1 protein expression in the photoaging research plays an important role. The most important composition of AP-1 protein are c-fos and c-jun.With development of studying on the anti-aging mechanism, researchers have found that the cytokine and the cytokine receptor plays a very important role in regulating skin-aging, and have devoted more attention to it's study. However, the study about using cytokines to anti-skin aging are inadequate.
In this study, we use fibroblast and UVA to establish photo-aging model.We use different dose of UVA iridiate fibroblast. Then we test cell proliferation activity by MTT. The expression of c-fos and c-jun detected by immunocytochemistry and Western Blot method. At the same time with different doses of EGFR inhibitors treat fibroblasts and use UVA 15 J/cm2 irradiate fibroblastd.Then we observe cell morphology and cell proliferation, detect the changes of c-fos and c-jun expression. We discuss whether or not EGFR Inhibitor inhibits the expression of c-fos and c-jun and protect fibroblasts from UVA.
【Methods】
1. We culture fibroblasts of foreskin from young adults and using the five generation cells for experiment. We divide cells into four groups and use different doses of UVA (0 J/cm~2,5 J/cm~2,10J/cm~2,20J/cm~2) irradiate cells. Then we test cell proliferation activity by MTT, and use immunocytochemistry method to test expression of c-fos and c-jun protein in fibroblast.
2. We use the five generetion fibroblasts to do experiment and divided to five groups. By adding different doses of EGFR Inhibitor, small dose (0.1 ug/ml), medium dose (1.0 ug/ml), high dose (10.0 ug/ml) to the cultured fibroblasts, and use UVA15J/cm~2 irradiation.Then we test cell proliferation activity.by MTT, use immunocytochemistry and Western blot method to test expression of c-fos and c-jun protein in fibroblasts.
【Results】
1. Different doses of UVA (the small dose 5 J/cm2, the medium dose 10J/cm~2, high-dose 20J/cm~2) irradiation skin fibroblasts in vitro showes that:with the UVA radiation dose increased, fibroblast OD values decreased. Comparing the middle and high doses group with control group, the OD values have a very significant difference (p<0.01). The small dose group have a significant difference (p<0.05). Using different doses of UVA irradiate cells, the cells shape became round, the gap between cells increased, lose the original shuttle shape. Immunocytochemical result showes that:with the UVA radiation dose increased, c-fos, c-jun were positive in the cytoplasm and nucleus, OD values increased with the dose increased.Compared with control group, UVA 10 J/cm`2, UVA 20 J/cm~2 irradiation c-fos and c-jun expression was significantly different (P<0.05),5 J/cm~2 dose group has not significantly different (P>0.05).
2.We treat the fibroblast with different doses of EGFR inhibitors, small dose(0. 1ug/ml), medium dose (1.0 ug/ml), high-dose (10.0 ug/ml) and irradiated by UVA15 J/cm2. The results of MTT show that:with the EGFR inhibitor increase, OD values of fibroblasts increased compared with irradiation group, but have not statistically significance(P>0.05). It had not significant effect on cell morphology. Immunocytochemistry showes that: c-fos staining is positive in the cytoplasm and nucleus, OD values decreased with EGFR inhibitor increased. But the c-jun expression have not statistically significant between experiment group and the control group(P>0.05). The Western blot results showed that:for c-fos and c-jun protein, compared with the control group, medium and high-dose group have statistically significant (P<0.05).
【Conclusion】
1. Cultured skin fibroblasts, UVA radiation cause fibroblast aging, the proliferation decreased, and the shape changed from the spindle to round cells, while AP-1 activation of intracellular protein (c-fos, c-jun) highly expressed.
2. We treat the fibroblast with different doses of EGFR inhibitors and irradiated by UVA15 J/cm2.This could not improve skin fibroblast proliferation activity in vitro, and no change in cells'morphology. And after treated by EGFR inhibitors, intracellular c-fos, c-jun expression decreased. This will induce the synthesis of MMPs and keep the balance of collagen.This protect the fibroblasts from the photoaging of UVA.
引文
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