17-β雌二醇对人皮肤成纤维细胞产生透明质酸和胶原的影响
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摘要
目的：诸多研究表明女性更年期后皮肤老化加速可能与雌激素减少造成透明质酸和胶原水平降低有关,本研究旨在探讨17-β雌二醇对体外培养人皮肤成纤维细胞产生透明质酸和胶原的影响,明确更年期后皮肤老化加速可能与雌激素的关系。
方法：将人皮肤成纤维细胞接种于无酚红DMEM培养基培养,待细胞培养至接近融合,加入浓度分别为：1×10-11 mol/L、1×10-10 mol/L、1×10-9mol/L、1×10-8mol/L的17-B雌二醇,并设不加药的空白组作为对照,每组设6个复孔,48小时后用酶联免疫法检测培养液中透明质酸含量。碱水解法检测培养液中羟脯氨酸含量,以之推测胶原合成能力。
结果：对照组、1×10-11 mol/L、1×10-10 mol/L17-β雌二醇浓度组透明质酸含量两两比较均无差别(P>0.05),1×10-9mol/L17-β雌二醇浓度组较对照组透明质酸浓度高,差异有显著统计学意义(P<0.01),1×10-8mol/L17-β雌二醇浓度组较对照组高,差异有统计学意义(P<0.05),但1×10-9mol/L组与1×10-8mol/L组相比差异无统计学意义(P>0.05)；1×10-11mol/L与1×10-10mol/L组羟脯氨酸浓度差异有统计学意义(P<0.05),对照组、1×10-11mol/L、1×10-10mol/L、1×10-9mol/L17-β雌二醇浓度组羟脯氨酸含量均依次增加,差异均有显著统计学意义(P<0.01)；1×10-8mol/L浓度组羟脯氨酸含量较1×10-9mol/L浓度组低,差异有显著统计学意义(P<0.01)；培养上清中的透明质酸与羟脯氨酸浓度呈正相关关系(r=0.794,P<0.01)。
结论：17-β雌二醇高于一定浓度时对人皮肤成纤维细胞产生透明质酸具有促进作用,但可能并无浓度依赖性。当17-β雌二醇浓度小于1×10-9mol/L时,其对皮肤成纤维细胞产生胶原具有浓度依赖性促进作用,但当其浓度再增加时,可能17-β雌二醇促进胶原产生的作用降低。透明质酸可能影响胶原的产生。更年期后女性雌激素水平降低造成真皮成纤维细胞透明质酸和胶原生成减少可能是皮肤老化加速的重要原因,雌激素干预可能对延缓女性皮肤老化有益。
Objective Many reports have showed that skin aging after menopause in female may have relationship with the decrease of hyaluronic acid (HA) and collagen level. This report aims to explore the effects of 17-βestradiol on hyaluronic acid and collagen synthesis in cultured human skin fibroblasts.
Methods The human skin fibroblasts were cultured in DMEM medium without phenol red in vitro. Serial dosages of 17-βestradiol were added to the culture system. Then the fibroblasts were divided into five groups according to the dosage of 17-βestradiol. They were control group, 1×10-11, 1×10-10, 1×10-9, 1×10-8mol/L 17-βestradiol group with 6 units in each group. After 48 hours the hyaluronic acid concentration in cell culture fluid was determined by ELISA method. The hydroxyproline concentration was determined by alkaline hydrolysis method to speculate the collagen synthesis ability.
Results In the control group,10-11mol/L and 10-10mol/L 17-βestradiol groups the HA concentrations had no statistical significance (p>0.05). The HA concentration of 10-9 mol/L 17-βestradiol groups was higher than that of control group which had notable statistical significance (p<0.01). The HA concentration of 10-8 mol/L 17-βestradiol group was higher than that of control group which had statistical significance (p<0.05). But the difference of HA concentrations between 10-9 mol/L and 10-8 mol/L 17-βestradiol groups had no statistical significance (p>0.05). The difference of hydroxyproline concentration between 1×10-11 and 1×10-10 mol/L groups had statistical significance (p<0.05), in control group and 1×10-11mol/L, 1×10-10mol/L, 1×10-9mol/L 17-βestradiol groups the hydroxyproline concentration of the later group was higher than that of the former and the differences all had notable statistical significance (P<0.01). The hydroxyproline concentration of the 1×10-8 mol/L group is lower than that of the 1×10-9 mol/L group. The concentrations of HA and collagen showed positive correlation remarkably (r=0.794, p<0.01).
Conclusions 17-Bestradiol could promote HA synthesis when its concentration is higher than some level. But the promotional effect may doesn't depend on the concentration of 17-Bestradiol. Hydroxyproline is highly special amino acid with constant content in collagen, then in general the hydroxyproline concentration could reflect collagen concentration. So the promotional effect of 17-Bestradiol on collagen synthesis in cultured human skin fibroblasts depends on its concentration when its concentration below 10-9 mol/L. But when its concentration is higher than 10-9 mol/1 the promotional effect may decrease. HA may influence the production of collagen. The influence of the change of estrogen level to the HA and collagen production in fibroblasts may be an important factor to accelerate skin aging in postmenopausal women. Estrogen may be a beneficial factor to delay skin aging in female.
方法：将人皮肤成纤维细胞接种于无酚红DMEM培养基培养,待细胞培养至接近融合,加入浓度分别为：1×10-11 mol/L、1×10-10 mol/L、1×10-9mol/L、1×10-8mol/L的17-B雌二醇,并设不加药的空白组作为对照,每组设6个复孔,48小时后用酶联免疫法检测培养液中透明质酸含量。碱水解法检测培养液中羟脯氨酸含量,以之推测胶原合成能力。
结果：对照组、1×10-11 mol/L、1×10-10 mol/L17-β雌二醇浓度组透明质酸含量两两比较均无差别(P>0.05),1×10-9mol/L17-β雌二醇浓度组较对照组透明质酸浓度高,差异有显著统计学意义(P<0.01),1×10-8mol/L17-β雌二醇浓度组较对照组高,差异有统计学意义(P<0.05),但1×10-9mol/L组与1×10-8mol/L组相比差异无统计学意义(P>0.05)；1×10-11mol/L与1×10-10mol/L组羟脯氨酸浓度差异有统计学意义(P<0.05),对照组、1×10-11mol/L、1×10-10mol/L、1×10-9mol/L17-β雌二醇浓度组羟脯氨酸含量均依次增加,差异均有显著统计学意义(P<0.01)；1×10-8mol/L浓度组羟脯氨酸含量较1×10-9mol/L浓度组低,差异有显著统计学意义(P<0.01)；培养上清中的透明质酸与羟脯氨酸浓度呈正相关关系(r=0.794,P<0.01)。
结论：17-β雌二醇高于一定浓度时对人皮肤成纤维细胞产生透明质酸具有促进作用,但可能并无浓度依赖性。当17-β雌二醇浓度小于1×10-9mol/L时,其对皮肤成纤维细胞产生胶原具有浓度依赖性促进作用,但当其浓度再增加时,可能17-β雌二醇促进胶原产生的作用降低。透明质酸可能影响胶原的产生。更年期后女性雌激素水平降低造成真皮成纤维细胞透明质酸和胶原生成减少可能是皮肤老化加速的重要原因,雌激素干预可能对延缓女性皮肤老化有益。
Objective Many reports have showed that skin aging after menopause in female may have relationship with the decrease of hyaluronic acid (HA) and collagen level. This report aims to explore the effects of 17-βestradiol on hyaluronic acid and collagen synthesis in cultured human skin fibroblasts.
Methods The human skin fibroblasts were cultured in DMEM medium without phenol red in vitro. Serial dosages of 17-βestradiol were added to the culture system. Then the fibroblasts were divided into five groups according to the dosage of 17-βestradiol. They were control group, 1×10-11, 1×10-10, 1×10-9, 1×10-8mol/L 17-βestradiol group with 6 units in each group. After 48 hours the hyaluronic acid concentration in cell culture fluid was determined by ELISA method. The hydroxyproline concentration was determined by alkaline hydrolysis method to speculate the collagen synthesis ability.
Results In the control group,10-11mol/L and 10-10mol/L 17-βestradiol groups the HA concentrations had no statistical significance (p>0.05). The HA concentration of 10-9 mol/L 17-βestradiol groups was higher than that of control group which had notable statistical significance (p<0.01). The HA concentration of 10-8 mol/L 17-βestradiol group was higher than that of control group which had statistical significance (p<0.05). But the difference of HA concentrations between 10-9 mol/L and 10-8 mol/L 17-βestradiol groups had no statistical significance (p>0.05). The difference of hydroxyproline concentration between 1×10-11 and 1×10-10 mol/L groups had statistical significance (p<0.05), in control group and 1×10-11mol/L, 1×10-10mol/L, 1×10-9mol/L 17-βestradiol groups the hydroxyproline concentration of the later group was higher than that of the former and the differences all had notable statistical significance (P<0.01). The hydroxyproline concentration of the 1×10-8 mol/L group is lower than that of the 1×10-9 mol/L group. The concentrations of HA and collagen showed positive correlation remarkably (r=0.794, p<0.01).
Conclusions 17-Bestradiol could promote HA synthesis when its concentration is higher than some level. But the promotional effect may doesn't depend on the concentration of 17-Bestradiol. Hydroxyproline is highly special amino acid with constant content in collagen, then in general the hydroxyproline concentration could reflect collagen concentration. So the promotional effect of 17-Bestradiol on collagen synthesis in cultured human skin fibroblasts depends on its concentration when its concentration below 10-9 mol/L. But when its concentration is higher than 10-9 mol/1 the promotional effect may decrease. HA may influence the production of collagen. The influence of the change of estrogen level to the HA and collagen production in fibroblasts may be an important factor to accelerate skin aging in postmenopausal women. Estrogen may be a beneficial factor to delay skin aging in female.
引文
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2. Zhang W, Watson CE, Liu C, et al. Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in dermal fibroblasts and in osteoblasts:a molecular mechanism contributing to organ atrophy. Biochem J,2000,349(1):91-97.
3. Sugiyama Y, Shimada A, Sayo T, et al. Putative hyaluronan synthase mRNA are expressed in mouse skin and TGF-beta upregulates their expression in cultured human skin cells. J Invest Dermatol,1998,110(2):116-121.
4. Stern R, Maibach HI. Hyaluronan in skin:aspects of aging and its pharmacologic modulation. Clin Dermatol,2008,26(2):106-122.
5. Thomas BF, Irwin MF, Arthur ZE, et al. Fitzpatrick's dermatology in general medicine.6th ed. New York:McGraw-Hill,2003:165.
6. Haczynski J, Tarkowski R, Jarzabek K, et al. Human cultured skin fibroblasts express estrogen receptor alpha and beta. Int J Mol Med,2002,10(2):149-153.
7.李晓凤,范卫新,张卉.苯甲酸雌二醇及UVA辐照对体外培养的人皮肤成纤维细胞胶原合成的影响.中华皮肤科杂志,2006,39(7)：385-387.
8. Emma P, Nungavarm R, Paivi M, et al. Would healing in ovariectomizderats:effects of chemically modified tetracycline (CMT-8) and estrogen on matrix metalloproteinases-8,-13 and type I collagen express. Curr Med Chem,2001,8(3):281-294.
9. Son ED, Lee JY, Lee S, et al. Topical application of 17beta-estradiol increases extracellular matrix protein synthesis by stimulating tgf-Beta signaling in aged human skin in vivo. J Invest Dermatol,2005,124(6):1149-1161.
10.李天航,刘原君,范丽云等.雌二醇对体外培养的人皮肤成纤维细胞基质蛋白酶-1基因表达的影响.中华皮肤科杂志,2008,41(7)：481-482.
11. Waller JM, Maibach HI. Age and skin structure and function, a quantitative approach (Ⅱ):protein, glycosaminoglycan, water, and lipid content and structure. Skin Res Technol,2006,12(3):145-154.
12. Dunn LB, Damesyn M, Moore AA, et al. Does estrogen prevent skin aging? Results from the First National Health and Nutrition Examination Survey (NHANES I). Arch Dermatol,1997,133:339-342.
13. Schmidt JB, Binder M, Demschik G, et al. Treatment of skin aging with topical estrogens. Int J Dermatol,1996,35(9):669-674.
14. Affinito P, Palomba S, Sorrentino C, et al. Effects of postmenopausal hypoestrogenism on skin collagen. Maturitas,1999,33(3):239-247.
15. Nawrat P, Surazynski A, Karna E,et al. The effect of hyaluronic acid on interleukin-1-induced deregulation of collagen metabolism in cultured human skin fibroblasts. Pharmacol Res,2005,51(5):473-477.
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2. Zhang W, Watson CE, Liu C, et al. Glucocorticoids induce a near-total suppression of hyaluronan synthase mRNA in dermal fibroblasts and in osteoblasts:a molecular mechanism contributing to organ atrophy. Biochem J,2000,349(1):91-97.
3. Sugiyama Y, Shimada A, Sayo T, et al. Putative hyaluronan synthase mRNA are expressed in mouse skin and TGF-beta upregulates their expression in cultured human skin cells. J Invest Dermatol,1998,110(2):116-121.
4. Stern R, Maibach HI. Hyaluronan in skin:aspects of aging and its pharmacologic modulation. Clin Dermatol,2008,26(2):106-122.
5. Thomas BF, Irwin MF, Arthur ZE, et al. Fitzpatrick's dermatology in general medicine.6th ed. New York:McGraw-Hill,2003:165.
6. Haczynski J, Tarkowski R, Jarzabek K, et al. Human cultured skin fibroblasts express estrogen receptor alpha and beta. Int J Mol Med,2002,10(2):149-153.
7.李晓凤,范卫新,张卉.苯甲酸雌二醇及UVA辐照对体外培养的人皮肤成纤维细胞胶原合成的影响.中华皮肤科杂志,2006,39(7)：385-387.
8. Emma P, Nungavarm R, Paivi M, et al. Would healing in ovariectomizderats:effects of chemically modified tetracycline (CMT-8) and estrogen on matrix metalloproteinases-8,-13 and type I collagen express. Curr Med Chem,2001,8(3):281-294.
9. Son ED, Lee JY, Lee S, et al. Topical application of 17beta-estradiol increases extracellular matrix protein synthesis by stimulating tgf-Beta signaling in aged human skin in vivo. J Invest Dermatol,2005,124(6):1149-1161.
10.李天航,刘原君,范丽云等.雌二醇对体外培养的人皮肤成纤维细胞基质蛋白酶-1基因表达的影响.中华皮肤科杂志,2008,41(7)：481-482.
11. Waller JM, Maibach HI. Age and skin structure and function, a quantitative approach (Ⅱ):protein, glycosaminoglycan, water, and lipid content and structure. Skin Res Technol,2006,12(3):145-154.
12. Dunn LB, Damesyn M, Moore AA, et al. Does estrogen prevent skin aging? Results from the First National Health and Nutrition Examination Survey (NHANES I). Arch Dermatol,1997,133:339-342.
13. Schmidt JB, Binder M, Demschik G, et al. Treatment of skin aging with topical estrogens. Int J Dermatol,1996,35(9):669-674.
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