N-乙酰葡萄糖胺对皮肤角质形成细胞透明质酸合成的影响
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摘要
背景和目的:透明质酸(Hyaluronic acid,HA)是天然存在于人体皮肤中的具有保水功能的聚合物,由葡糖醛酸和N-乙酰葡萄糖胺(又称N-乙酰氨基葡萄糖,N-Acetylglucosamine, NAG)组成直链高分子多糖,作为吸水的大分子位于细胞外基质(Extracellular matrix, ECM),广泛存在于脊椎动物结缔组织和某些细菌内。HA分子在溶液中高度伸展和随机卷曲构型使其占有很大区域,其流体动力学体积较未水化时所占空间大l0倍,而且分子链之间相互缠绕形成连续的网状结构,水分子通过极性键和氢键与其直接作用。使得HA像“分子海绵”一样,可以吸收和保持其自身重量上千倍的水分,是国际上公认的最好的保湿剂之一,具有维持细胞外间隙,并促进离子溶质和营养物质的运输。除此之外,HA还可与蛋白结合成为分子量更大的蛋白多糖分子,分布在结缔组织中的蛋白多糖,通过氨基多糖与水结合,这种HA-蛋白质-水形成的凝胶将细胞粘合在一起,发挥正常的细胞代谢作用及组织保水作用,并保护细胞不受病原菌侵害,防止感染,使皮肤具有一定的坚韧性和弹性。年青健康的皮肤与含水量高密切相关,随着年龄的增长,皮肤中水分减少,进而弹性下降,光泽消退,皮肤粗糙衰老。研究表明真皮组织含水量与HA含量有直接关系。皮肤HA的含量在机体的器官中最高,占人体全部HA含量的50%。随着对HA生理功能的深入研究,HA作为化妆品成分用于保养皮肤倍受重视。
皮肤中的HA主要由皮肤角质形成细胞和成纤维细胞产生,而NAG为HA合成原料之一,故本实验以构成人表皮的主要细胞角质形成细胞(Human epidermal keratinocytes-adult, HEKa)作为切入点,研究外源性NAG对HEKa细胞HA合成的影响,及探讨NAG影响HEKa细胞HA合成的分子机制。
方法:采用四甲基偶氮唑蓝(MTT)法检测不同浓度NAG对HEKa细胞增殖的影响;酶联免疫吸附试验(Enzyme linked immunosorbent assay, ELISA)检测不同浓度NAG对HEKa细胞HA合成的影响;逆转录聚合酶链反应(Reverse transcription-polymerase chain raction,RT-PCR)测定NAG对HEKa细胞透明质酸合成酶(Hyaluronic acid synthetase, HAS)mRNA的影响,包括HAS1、HAS2和HAS3 mRNA,研究其作用的分子机制。
结果:MTT结果表明,低浓度的NAG(1mM/L、3mM/L和5mM/L)对HEKa细胞的增殖抑制无明显影响(P>0.05),而10 mM/L的NAG显著抑制HEKa细胞的增殖(P<0.05);ELISA结果显示,NAG呈剂量依赖性促进HEKa细胞HA的生成(P<0.01),最佳浓度为5mM/L;RT-PCR结果显示,NAG对细胞HAS1和HAS2mRNA的表达无明显作用,而1mM/L和5mM/LNAG上调HAS3mRNA的表达,10mM/L的NAG对HAS3mRNA的表达无明显影响。
结论:(1)不同浓度NAG作用于HEKa,可促进HEKa细胞HA的合成和分泌。(2)高浓度NAG对HEKa细胞增殖具有抑制作用,而低浓度无影响,表明NAG不是通过促进细胞增殖而促进HEKa细胞HA的合成和分泌。其抑制作用有待于进一步研究。(3)NAG促进HEKa细胞HA合成,可能通过上调HAS3mRNA水平或增加HAS活性来增加HA合成,同时有可能作为原料增加而促进HA合成。(4)NAG作为化妆品的成分具有很好的开发前景。
Backgrounds and objectives: Hyaluronic acid (HA) is the linear chain giant molecular polycose composed of glucuronic Acid and N-Acetyl glucosamine (NAG), the polymer of keeping water which exists in the human skin. It is situated in extracellular matrix(ECM) as a water-absorbing macromolecule, and generally exists in vertebrate connective tissue and some bacteria. The strong extension and random curling of HA molecule result in tremendous domain, the hydrodynamics volume is ten times larger than the possessing space of its un-hydrated form. Further more, molecule chains twist between each other to form a consecutive reticular structure, which reacts with water molecule via polar bond and hydrogen bond. HA can absorb and keep water weighing up to thousand fold more than itself because of its structure like“molecule sponge”, and is one of the best internationally accepted moisturizers , keeping extracellular space and promoting ion disolving and transportation of nutrient substances. Besides, proteoglycan composed of HA and protein , has the large molecular weight. Proteoglycan binding to water through glucidamin, exists in connective tissue. The cells are binded by the gelatum composed of HA-protein-water, to run normally the functions of cell metabolism and tissue keeping water, to protect themselves from pathogenic bacteria and prevent infection, and to keep tenacity and elasticity of the skin. Youthful and healthy skin is closely related to its high moisture capacity, which decreased as the age increased. And then the skin elasticity decreases, the gloss fades away and the skin become rough and aging. The previous study indicated that the moisture capacity of corium is related to HA content. The content of skin HA, comprised 50% of the total content, is the highest in the body. With in-depth study on the physiologic function of HA, HA applyingin skin care has received wide attention as a cosmetic ingredient.
HA is mainly produced by human epidermal keratinocytes and dermal fibroblasts in skin, and NAG is one of the materials for HA synthesis.The present experiment is focused on adult human epidermal keratinocytes (HEKa), in order to study the effects of exogenous NAG on HA production in HEKa cells and investigate the molecular mechanism of the HA synthesis in HEKa cells affected by NAG.
Methods: The cell proliferation of HEKa cells affected by NAG were measured by MTT method. Effects of NAG at different concentrations on HA production in HEKa cells were measured by Enzyme linked immunosorbent assay (ELISA). Effects of NAG on Hyaluronic acid synthetase(HAS)mRNA in HEKa cells , including HAS1, HAS2 and HAS3 mRNA, were measured by Reverse transcription-polymerase chain raction (RT-PCR), in order to study the molecular mechanism of NAG.
Results: The result of MTT indicated that NAG at 1mM/L, 3mM/L and 5mM/L had no effect on cell proliferation of HEKa cells(P>0.05), but NAG at 10 mM/L significantly inhibited the cell proliferation of HEKa cells (P<0.05). The result of ELISA showed that NAG promoting HA production in HEKa cells in dose dependence(P<0.01),and the best concentration was 5mM/L. The result of RT-PCR showed that NAG had no effect on the expression of HAS1 and HAS2 mRNA in HEKa cells, however, NAG at 1mM/L and 5mM/L upregulated the expression of HAS3 mRNA. NAG at 10mM/L had no effect on the expression of HAS3mRNA.
Conclusions:(1)NAG at different concentrations can promoting HA production and secretion.(2)NAG at high concentration inhibits the cell proliferation of HEKa cells, but there are no effect at lower concentrations, which show that the effect of promoting HA production and secretion is not through cell proliferation. However, the inhibitive effect needs further study.(3)NAG may up-regulate the level of HAS3 mRNA and increase the activity, and may act as material to promoting HA production in HEKa cells. (4)There is good development prospect of NAG as a cosmetic ingredient.
皮肤中的HA主要由皮肤角质形成细胞和成纤维细胞产生,而NAG为HA合成原料之一,故本实验以构成人表皮的主要细胞角质形成细胞(Human epidermal keratinocytes-adult, HEKa)作为切入点,研究外源性NAG对HEKa细胞HA合成的影响,及探讨NAG影响HEKa细胞HA合成的分子机制。
方法:采用四甲基偶氮唑蓝(MTT)法检测不同浓度NAG对HEKa细胞增殖的影响;酶联免疫吸附试验(Enzyme linked immunosorbent assay, ELISA)检测不同浓度NAG对HEKa细胞HA合成的影响;逆转录聚合酶链反应(Reverse transcription-polymerase chain raction,RT-PCR)测定NAG对HEKa细胞透明质酸合成酶(Hyaluronic acid synthetase, HAS)mRNA的影响,包括HAS1、HAS2和HAS3 mRNA,研究其作用的分子机制。
结果:MTT结果表明,低浓度的NAG(1mM/L、3mM/L和5mM/L)对HEKa细胞的增殖抑制无明显影响(P>0.05),而10 mM/L的NAG显著抑制HEKa细胞的增殖(P<0.05);ELISA结果显示,NAG呈剂量依赖性促进HEKa细胞HA的生成(P<0.01),最佳浓度为5mM/L;RT-PCR结果显示,NAG对细胞HAS1和HAS2mRNA的表达无明显作用,而1mM/L和5mM/LNAG上调HAS3mRNA的表达,10mM/L的NAG对HAS3mRNA的表达无明显影响。
结论:(1)不同浓度NAG作用于HEKa,可促进HEKa细胞HA的合成和分泌。(2)高浓度NAG对HEKa细胞增殖具有抑制作用,而低浓度无影响,表明NAG不是通过促进细胞增殖而促进HEKa细胞HA的合成和分泌。其抑制作用有待于进一步研究。(3)NAG促进HEKa细胞HA合成,可能通过上调HAS3mRNA水平或增加HAS活性来增加HA合成,同时有可能作为原料增加而促进HA合成。(4)NAG作为化妆品的成分具有很好的开发前景。
Backgrounds and objectives: Hyaluronic acid (HA) is the linear chain giant molecular polycose composed of glucuronic Acid and N-Acetyl glucosamine (NAG), the polymer of keeping water which exists in the human skin. It is situated in extracellular matrix(ECM) as a water-absorbing macromolecule, and generally exists in vertebrate connective tissue and some bacteria. The strong extension and random curling of HA molecule result in tremendous domain, the hydrodynamics volume is ten times larger than the possessing space of its un-hydrated form. Further more, molecule chains twist between each other to form a consecutive reticular structure, which reacts with water molecule via polar bond and hydrogen bond. HA can absorb and keep water weighing up to thousand fold more than itself because of its structure like“molecule sponge”, and is one of the best internationally accepted moisturizers , keeping extracellular space and promoting ion disolving and transportation of nutrient substances. Besides, proteoglycan composed of HA and protein , has the large molecular weight. Proteoglycan binding to water through glucidamin, exists in connective tissue. The cells are binded by the gelatum composed of HA-protein-water, to run normally the functions of cell metabolism and tissue keeping water, to protect themselves from pathogenic bacteria and prevent infection, and to keep tenacity and elasticity of the skin. Youthful and healthy skin is closely related to its high moisture capacity, which decreased as the age increased. And then the skin elasticity decreases, the gloss fades away and the skin become rough and aging. The previous study indicated that the moisture capacity of corium is related to HA content. The content of skin HA, comprised 50% of the total content, is the highest in the body. With in-depth study on the physiologic function of HA, HA applyingin skin care has received wide attention as a cosmetic ingredient.
HA is mainly produced by human epidermal keratinocytes and dermal fibroblasts in skin, and NAG is one of the materials for HA synthesis.The present experiment is focused on adult human epidermal keratinocytes (HEKa), in order to study the effects of exogenous NAG on HA production in HEKa cells and investigate the molecular mechanism of the HA synthesis in HEKa cells affected by NAG.
Methods: The cell proliferation of HEKa cells affected by NAG were measured by MTT method. Effects of NAG at different concentrations on HA production in HEKa cells were measured by Enzyme linked immunosorbent assay (ELISA). Effects of NAG on Hyaluronic acid synthetase(HAS)mRNA in HEKa cells , including HAS1, HAS2 and HAS3 mRNA, were measured by Reverse transcription-polymerase chain raction (RT-PCR), in order to study the molecular mechanism of NAG.
Results: The result of MTT indicated that NAG at 1mM/L, 3mM/L and 5mM/L had no effect on cell proliferation of HEKa cells(P>0.05), but NAG at 10 mM/L significantly inhibited the cell proliferation of HEKa cells (P<0.05). The result of ELISA showed that NAG promoting HA production in HEKa cells in dose dependence(P<0.01),and the best concentration was 5mM/L. The result of RT-PCR showed that NAG had no effect on the expression of HAS1 and HAS2 mRNA in HEKa cells, however, NAG at 1mM/L and 5mM/L upregulated the expression of HAS3 mRNA. NAG at 10mM/L had no effect on the expression of HAS3mRNA.
Conclusions:(1)NAG at different concentrations can promoting HA production and secretion.(2)NAG at high concentration inhibits the cell proliferation of HEKa cells, but there are no effect at lower concentrations, which show that the effect of promoting HA production and secretion is not through cell proliferation. However, the inhibitive effect needs further study.(3)NAG may up-regulate the level of HAS3 mRNA and increase the activity, and may act as material to promoting HA production in HEKa cells. (4)There is good development prospect of NAG as a cosmetic ingredient.
引文
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46.王敬旭 .透明质酸及透明质酸酶在肿瘤诊断中的研究进展.国外医学肿瘤分册,2004;31:752—754。
47.Bergeret G C,Latouche X,Illouz Y G.The value of a fill ermaterial in corrective and cosmetics urgery:Derma Live and Derma Deep.Aesthetic Plast Surg, 2001; 25: 249-255.
48.Collawn SS. New product for treating facial wrinkles. FDAConsumer,2004;38:2.
49 . Yamamoto H . Antiaging and cosmetic effects of dietary hyaluronie acid (extracellular matrix extract).New Food lnd, 1998;40:33-41.
50.马振友等.皮肤美容化妆品制剂手册. 中医古籍出版社, 2004,512-620。
51.Moseley R, Leaver M, Walker M, et al. Comparison of the antioxidant properties of HYAFF-11p75, AQUACEL and hyaluronan towards reactive oxygen species in vitro. Biomaterials, 2002; 23:2255-2264.
52.Iordanov I, Bainova A, Chipilska L. A hygienic study of new raw materials for cosmetic agents and household chemical preparations. Probl Khig, 1990; 15: 30-38.