TGF-β/Smad信号转导通路对紫外线致人工皮肤光老化中MMP-1、pro-collagenⅠ mRNA表达影响的研究
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摘要
目的：
通过体外构建人工皮肤器官型培养基质金属蛋白酶1、Ⅰ型前胶原蛋白、TGF-βⅠ型受体及TGF-βⅡ型受体mRN,建立人工皮肤光老化模型,检测紫外线致人工皮肤光老化中A的表达作用,探讨TGF-β／Smad信号转导通路在皮肤光老化中的作用,为光保护剂的开发提供理论依据及研究靶点。
方法：
1.通过三维器官培养模型制备含有表皮及真皮的人工皮肤替代物；
2.以不同剂量UV(波长280-400nm,0J/cm2-40J/cm2)照射人皮肤成纤维细胞,通过MTT法检测细胞的活性,筛选建立人工皮肤光老化模型的紫外线剂量；
3.通过Real-Time RT-PCR法(荧光染料掺入法)检测紫外线致人工皮肤光老化模型中基质金属蛋白酶1、Ⅰ型前胶原蛋白、TGFβRⅠ及TGFβRⅡmRNA表达量的变化。
结果：
第一部分：于体外成功构建含有表皮、真皮的复合人工皮肤,HE染色真皮层可见排列呈网状的胶原纤维,其间为均匀分布的成纤维细胞,表皮层可见分层排列的角质形成细胞；
第二部分：MTT结果显示与空白组相比,10J/cm2和20J/cm2UV照射人工皮肤时细胞活性未受到明显的抑制,大于等于30J／cm2的照射剂量使细胞活性明显下降,呈剂量依赖性；
第三部分：(1)30 J/cm2紫外线照射人工皮肤后,基质金属蛋白酶1的mRNA表达量明显增加(P<0.05)；加入TGF-β1后表达量显著下调(P<0.01)；而UV照射后8小时加入TGF-β1,该基因mRNA的表达量仍较对照组高(P<0.05)；
(2)30 J/cm2紫外线明显抑制Ⅰ型前胶原蛋白mRNA的表达(P<0.05)；加入TGF-β1后表达量显著增高(P<0.01)；而UV照射后8小时加入TGF-β1,该基因mRNA的表达量仍较对照组低(P<0.05),差异有统计学意义；
(3)TGFβRⅠ的mRNA表达在不同处理组间无显著性差异(P>0.05)；
(4)30 J/cm2紫外线照射人工皮肤后,TGFβRⅡmRNA的表达量显著下调(P<0.01)；加入TGF-β1后表达量显著增加(P<0.01)；而UV照射后8小时加入TGF-β1,该基因mRNA的表达量仍较对照组低(P<0.01),差异有统计学意义。
结论：
1.成功构建含有表皮及真皮的复合人工皮肤；
2.首次应用复合人工皮肤建立光老化模型；
3.证实UV可以抑制人工皮肤中Ⅰ型前胶原mRNA的表达,同时上调MMP-1的表达,促使人工皮肤中真皮胶原减少,而这种调控作用与TGF-βRⅡ的表达受到抑制,TGF-β/Smad信号传导通路被削弱有关；
4.证实TGF-βRⅠ在紫外线致人工皮肤光老化过程中无明显作用。
Objectives:1. to construct the photoaging model of artificial skin by culturing and constructing artificial skin in vitro.2. to study the effect of MMP-1, pro-collagenⅠ, TGF-βtypeⅠand TGF-βtypeⅡreceptors mRNA expression of artificial skin photoaging by solar ultraviolet.3. to investigate the effect of TGF-β/Smad Signal Transduction Pathway in the process of skin photoaging.
Methods:1. The compound artificial skin including epidermis and dermis was prepared by culturing 3D organ cultivation model.2. The activity of fibroblast cells, which were irradiated by UV(wavelength of 280-400nm,0J/cm2-40J/cm2), were tested by MTT assay, to choose a proper UV dose.3. The changes of mRNA expression quantity of MMP-1, pro-collagenⅠ, TGF-βtypeⅠand TGF-βtypeⅡreceptors were tested by means of Real-Time RT-PCR (SYBR GreenⅠDNA-binding dye).
Results:
PartⅠ:The compound artificial skin including epidermis and dermis was constructed successfully. Fibroblast cells, which distribute in reticular collagen fibers, and layered keratinocyte were reticulate observed by HE staining.
PartⅡ:Comparing with blank group, the viability of cells in artificial skin, which was irradiated by 10J/cm2 UV or 20J/cm2 UV, were not inhibited obviously. However, when the dose of UV was up to or more than 30 J/cm2, the viability of cells in the treatment group decreases obviously in dose-dependent manner.
PartⅢ:1.After the artificial skin was irradiated by 30 J/cm2 UV, the amount of mRNA of MMP-1 increased obviously (P＜0.05); after added with TGF-β1, it decreased significantly (P＜0.01). Being added with TGF-β1 8 hours after irradiation of 30 J/cm2 UV, it was higher than the control (P＜0.05).
2. After being exposed by 30 J/cm2 UV, the mRNA expression quantity of pro-collagenⅠin artificial skin reduced obviously (P＜0.05). After added with TGF-β1, it increased significantly (P＜0.01). Being added with TGF-β1 8 hours after the UV radiation, it was lower than the control group(P＜0.05).
3. The difference of the amount of mRNA expression of TGFβRⅠamong different groups was not obviously(P＞0.05).
4. After being exposed by 30 J/cm2 UV, the quantity of mRNA of TGF-βRⅡwas down-regulated significantly (P＜0.01). After added with TGF-β1, it reduced significantly (P＜0.01). Being added with TGF-β1 8 hours after exposure of 30 J/cm2 UV, it was lower than the control (P＜0.01).
Conclusion:1. The artificial skin with epidermis and dermis was constructed successfully.
2. The photoaging model of compound artificial skin was constructed for the first time.
3. It is proved that UV could suppress TGF-β/Smad signal transduction pathway by inhibiting the expression of TGF-βRⅡmRNA. While decreaseing the expression of pro-collagenⅠmRNA in dermis, UV could increase the expression MMP-1 mRNA. With these effects, UV made collagen synthesis decreased and its degradation increased, which resulted in photoaging of artificial skin.
4. It is proved that TGF-βRI didn't play an important role in the process of artificial skin photoaging by UV.
通过体外构建人工皮肤器官型培养基质金属蛋白酶1、Ⅰ型前胶原蛋白、TGF-βⅠ型受体及TGF-βⅡ型受体mRN,建立人工皮肤光老化模型,检测紫外线致人工皮肤光老化中A的表达作用,探讨TGF-β／Smad信号转导通路在皮肤光老化中的作用,为光保护剂的开发提供理论依据及研究靶点。
方法：
1.通过三维器官培养模型制备含有表皮及真皮的人工皮肤替代物；
2.以不同剂量UV(波长280-400nm,0J/cm2-40J/cm2)照射人皮肤成纤维细胞,通过MTT法检测细胞的活性,筛选建立人工皮肤光老化模型的紫外线剂量；
3.通过Real-Time RT-PCR法(荧光染料掺入法)检测紫外线致人工皮肤光老化模型中基质金属蛋白酶1、Ⅰ型前胶原蛋白、TGFβRⅠ及TGFβRⅡmRNA表达量的变化。
结果：
第一部分：于体外成功构建含有表皮、真皮的复合人工皮肤,HE染色真皮层可见排列呈网状的胶原纤维,其间为均匀分布的成纤维细胞,表皮层可见分层排列的角质形成细胞；
第二部分：MTT结果显示与空白组相比,10J/cm2和20J/cm2UV照射人工皮肤时细胞活性未受到明显的抑制,大于等于30J／cm2的照射剂量使细胞活性明显下降,呈剂量依赖性；
第三部分：(1)30 J/cm2紫外线照射人工皮肤后,基质金属蛋白酶1的mRNA表达量明显增加(P<0.05)；加入TGF-β1后表达量显著下调(P<0.01)；而UV照射后8小时加入TGF-β1,该基因mRNA的表达量仍较对照组高(P<0.05)；
(2)30 J/cm2紫外线明显抑制Ⅰ型前胶原蛋白mRNA的表达(P<0.05)；加入TGF-β1后表达量显著增高(P<0.01)；而UV照射后8小时加入TGF-β1,该基因mRNA的表达量仍较对照组低(P<0.05),差异有统计学意义；
(3)TGFβRⅠ的mRNA表达在不同处理组间无显著性差异(P>0.05)；
(4)30 J/cm2紫外线照射人工皮肤后,TGFβRⅡmRNA的表达量显著下调(P<0.01)；加入TGF-β1后表达量显著增加(P<0.01)；而UV照射后8小时加入TGF-β1,该基因mRNA的表达量仍较对照组低(P<0.01),差异有统计学意义。
结论：
1.成功构建含有表皮及真皮的复合人工皮肤；
2.首次应用复合人工皮肤建立光老化模型；
3.证实UV可以抑制人工皮肤中Ⅰ型前胶原mRNA的表达,同时上调MMP-1的表达,促使人工皮肤中真皮胶原减少,而这种调控作用与TGF-βRⅡ的表达受到抑制,TGF-β/Smad信号传导通路被削弱有关；
4.证实TGF-βRⅠ在紫外线致人工皮肤光老化过程中无明显作用。
Objectives:1. to construct the photoaging model of artificial skin by culturing and constructing artificial skin in vitro.2. to study the effect of MMP-1, pro-collagenⅠ, TGF-βtypeⅠand TGF-βtypeⅡreceptors mRNA expression of artificial skin photoaging by solar ultraviolet.3. to investigate the effect of TGF-β/Smad Signal Transduction Pathway in the process of skin photoaging.
Methods:1. The compound artificial skin including epidermis and dermis was prepared by culturing 3D organ cultivation model.2. The activity of fibroblast cells, which were irradiated by UV(wavelength of 280-400nm,0J/cm2-40J/cm2), were tested by MTT assay, to choose a proper UV dose.3. The changes of mRNA expression quantity of MMP-1, pro-collagenⅠ, TGF-βtypeⅠand TGF-βtypeⅡreceptors were tested by means of Real-Time RT-PCR (SYBR GreenⅠDNA-binding dye).
Results:
PartⅠ:The compound artificial skin including epidermis and dermis was constructed successfully. Fibroblast cells, which distribute in reticular collagen fibers, and layered keratinocyte were reticulate observed by HE staining.
PartⅡ:Comparing with blank group, the viability of cells in artificial skin, which was irradiated by 10J/cm2 UV or 20J/cm2 UV, were not inhibited obviously. However, when the dose of UV was up to or more than 30 J/cm2, the viability of cells in the treatment group decreases obviously in dose-dependent manner.
PartⅢ:1.After the artificial skin was irradiated by 30 J/cm2 UV, the amount of mRNA of MMP-1 increased obviously (P＜0.05); after added with TGF-β1, it decreased significantly (P＜0.01). Being added with TGF-β1 8 hours after irradiation of 30 J/cm2 UV, it was higher than the control (P＜0.05).
2. After being exposed by 30 J/cm2 UV, the mRNA expression quantity of pro-collagenⅠin artificial skin reduced obviously (P＜0.05). After added with TGF-β1, it increased significantly (P＜0.01). Being added with TGF-β1 8 hours after the UV radiation, it was lower than the control group(P＜0.05).
3. The difference of the amount of mRNA expression of TGFβRⅠamong different groups was not obviously(P＞0.05).
4. After being exposed by 30 J/cm2 UV, the quantity of mRNA of TGF-βRⅡwas down-regulated significantly (P＜0.01). After added with TGF-β1, it reduced significantly (P＜0.01). Being added with TGF-β1 8 hours after exposure of 30 J/cm2 UV, it was lower than the control (P＜0.01).
Conclusion:1. The artificial skin with epidermis and dermis was constructed successfully.
2. The photoaging model of compound artificial skin was constructed for the first time.
3. It is proved that UV could suppress TGF-β/Smad signal transduction pathway by inhibiting the expression of TGF-βRⅡmRNA. While decreaseing the expression of pro-collagenⅠmRNA in dermis, UV could increase the expression MMP-1 mRNA. With these effects, UV made collagen synthesis decreased and its degradation increased, which resulted in photoaging of artificial skin.
4. It is proved that TGF-βRI didn't play an important role in the process of artificial skin photoaging by UV.
引文
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[2]Fisher GJ, Kang SW, Varani J, et al. Mechanisms of photoaging and chronological skin aging. Arch Dermatol,2002,138(11):1462-1470.
[3]Kang SW, Fisher GJ, Voorhees JJ. Photoaging and topical tretinoin:therapy, pathogenesis, and prevention. Arch Dermatol,1997,133(10):1280-1284.
[4]Pascual-Le Tallec L, Korwin-Zmijowska C, Adolphe M. Effects of simulated solar radiation on type Ⅰ and type Ⅲ collagens, collagenase (MMP-1) and stromelysin (MMP-3) gene expression in human dermal fibroblasts cultured in collagen gels. Journal of Photochemistry and Photobiology B:Biology,1998,42(3):226-232.
[5]Fisher GJ, Datta SC, Talwar HS, et al. Molecular basis of sun-induced premature skin ageing and retinoid antagonism. Nature,1996,379(6563):335-339.
[6]Hodge C, Liao J, Stofega M, et al. Growth Hormone Stimulates Phosphorylation and Activation of Elk-1 and Expression of c-fos, egr-1, and junB through Activation of Extracellular Signal-regulated Kinases 1 and 2[J]. Biol Chem,1998,273 (47): 31327-31336.
[7]Fisher GJ, Talwar HS, Lin J, et al. Retinoic acid inhibits induction of c-Jun protein by ultraviolet radiation that occurs subsequent to activation of mitogen-activated protein kinase pathways in human skin in vivo[J]. J Clin invest, 1998,101(6):1432-1440.
[8]Rosette C, Karin M. Ultraviolet light and osmotic stress; activation of the JNK cascade through multiple growth factor and cytokine receptors. Science,1996,274 (5290):1194-1197.
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