白藜芦醇对培养的人黑素细胞的影响
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摘要
近年来,随着人口老化、环境污染、全球气温上升,越来越多的人出现皮肤色素失调性疾病。寻找更安全、更有效的新型外用皮肤美白祛斑剂是当今化妆品的研究热点之一。
细胞因子与黑素细胞之间的关系已成为美白研究的另一重要方向。在皮肤,内皮素(endothelins, ET)表达于角质形成细胞,内皮素受体(endothelin receptor, EDNR)则表达于黑素细胞。已证实,ET可与黑素细胞膜上的内皮素受体相互作用并通过酪氨酸酶、酪氨酸酶相关蛋白-1(tyrosinase-related protein-1, TRP-1)、MAPK、PKC、PKA和酪氨酸激酶受体(receptor tyrosine kinase,KIT)这六条信号通路促进黑素细胞的增殖和黑素生成。因此,内皮素系统作为角质形成细胞和黑素细胞相互作用的桥梁,参与黑素细胞的增殖和黑素合成等过程,而内皮素拮抗剂可以通过拮抗ET的作用而抑制黑素细胞增殖及合成黑素。
已有报道证实白藜芦醇(resveratrol,RES)是一种非肽类内皮素拮抗剂。我们推测,RES作为一种内皮素拮抗剂可能从酪氨酸酶、TRP-1、MAPK、PKC、PKA和KIT这六条信号通路拮抗ET-1诱导的黑色素生成,从而达到美白祛斑的作用。
本课题主要目的是研究RES对ET-1介导的黑素细胞黑素合成的影响及其相关机制,并探讨其对黑素细胞的细胞毒性作用,为RES作为一种新型、安全和高效的皮肤美白祛斑物质提供一定的理论基础和实验依据。为了使该项研究顺利地完成,我们将整个实验细分为四个实验,分别是:①建立不含佛波醇酯(12-O-tetradecanoyl-phorbol-13-acetate,TPA)和霍乱毒素(cholera toxin,CT)体外培养正常人黑素细胞的方法:以避免TPA和CT改变黑素细胞的生物学特性,为后续的实验提供可靠的黑素细胞来源。②研究ET-1对培养的人黑素细胞生物学特性的影响:探讨ET-1对体外培养的正常人黑素细胞形态、增殖、酪氨酸酶活性及黑素合成的影响,并寻找一个最有效的调控浓度,为后续的实验提供实验基础。③RES对人黑素细胞的细胞毒性分析:以探讨RES应用于美白祛斑治疗的安全性,并寻找出其安全浓度范围,为其临床应用提供实验依据。④探讨RES对ET-1介导的黑素合成的影响及其相关机制:以探讨RES应用于美白祛斑治疗的有效性,并初步研究其相关机制,为其临床应用提供实验依据。针对以上四个实验目的,我们的实验方法和结果如下:
首先,在第一项实验研究中,我们取了青少年包皮环切术后包皮组织6例,年龄6-11岁,平均8岁,用不含TPA和CT的角质形成细胞无血清培养基(KC-SFM)培养、用差速贴壁法及胰酶差速消化法去除角质形成细胞、用两段法去除成纤维细胞,所培养出的细胞经多巴染色和抗S-100、抗HMB45蛋白抗体免疫细胞化学染色均呈阳性反应,证实为黑素细胞,从而建立了不含TPA和CT体外培养正常人黑素细胞的方法,该方法稳定、可靠。
其次,在第二项实验研究中,我们取了青少年包皮环切术后包皮组织6例,年龄6-15岁,平均11岁,分别用倒置显微镜观察、CCK-8法、多巴氧化法和NaOH法测定了ET-1对黑素细胞形态、增殖、酪氨酸酶活性和黑素含量的影响。该项实验结果提示:一定浓度的ET-1可有效地促进培养的正常人黑素细胞的增殖和树突形成,上调酪氨酸酶活性和增加黑素含量,10 nM的ET-1不仅能促进黑素细胞增殖及酪氨酸酶活性,而且此浓度下的黑素含量水平最高,因此10 nM是最有效的调控浓度。
再次,在分析RES对人黑素细胞的细胞毒性作用时,我们取了青少年包皮环切术后包皮组织3例,年龄6-12岁,平均10岁,用CCK-8法测定黑素细胞的增殖抑制率,Annexin V/PI双染色法流式细胞仪检测黑素细胞的凋亡率。该项实验结果显示,10μM浓度以下的RES对人黑素细胞无增殖抑制作用也无凋亡促进作用。
最后,在研究RES对ET-1介导的黑素合成的影响及其相关机制中,我们取了青少年包皮环切术后包皮组织9例,年龄6-15岁,平均12岁,采用NaOH法测定黑素含量,多巴氧化法测定黑素细胞的酪氨酸酶活性,半定量RT-PCR法测定酪氨酸酶mRNA和TRP-1 mRNA表达水平。我们的实验结果提示:0.1~10μM的RES可抑制ET-1介导的黑素合成,其主要机制可能是通过抑制酪氨酸酶活性起作用,而与酪氨酸酶mRNA和TRP-1mRNA的表达无关。
我们的实验结果表明,RES可作为一种内皮素拮抗剂,可以通过抑制酪氨酸酶活性减少黑素合成,且在10μM浓度以下时无细胞毒性作用。因此,RES可作为一种皮肤美白物质,其安全、有效的浓度范围是0.1~10μM。
In recent years, with the aging of population, environmental pollution, and global temperature rise, more and more people will develop pigmentation disorders, including age spots, melasma, chloasma, freckle, post inflammatory hyperpigmentation or sun-induced pigmented blemishes, etc. To discover the safer and more effective newer external-use skin lightening agents is now one of hot spots in the cosmetics research.
The relationship between cytokine and melanocyte has become another important research direction for the depigmenting agents. In epidermis, endothelins (ET) is expressed in keratinocytes and its receptor in melanocytes. It has been comfirmed that the ET can interact with its receptors located in melanocyte membrane to promote melanocyte proliferation and melanogenesis via six signal pathways including the tyrosinase, tyrosinase-related protein-1(TRP-1), MAPK, PKC, PKA and receptor tyrosine kinase(KIT) signal pathways. Thus, endothelins system, as a bridge connecting keratinocytes and melanocytes, participates in the proliferation and melanogenesis of melanocyte, and endothelin antagonist may inhibit cell proliferation and melanin synthesis of melanocytes through the antagonistic role of ET.
It has been comfirmed that resveratrol (RES) is a nonpeptide endothelin antagonist. So RES, acting as an endothelin antagonist, may efficiently antagonize the melanogenesis induced by ET-1 via six signal pathways including the tyrosinase, TRP-1, MAPK, PKC, PKA and KIT signal pathways.
The objective of this research project is to study the effects of RES on endothelin-1-induced melanogenesis and inhibitory mechanism, and explore cytotoxicity of RES on the normal cultured human melanocytes, to provide a theoretical basis and experimental evidenceand for development and application of RES as a novel, safe and highly effective skin lightening and depigmenting agent.The whole research project can be subdivided into the following four parts:①To establish a method of culturing melanocytes in the absence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and Cholera toxin(CT) : To avoid the oncogenicity of TPA and the change of biologic characteristics of cultured human melanocytes caused by adding TPA and CT, and provide the better melanocytes in biological characteristics for follow-up experiments.②To study effects of endothelin-1 on the biologic characteristics of cultured human melanocytes: To investigate the effects of endothelin-1 on the morphology, proliferation, tyrosinase activity and melanogenesis of normal human melanocytes cultured in vitro, and find one of the most effective regulatory concentration of ET-1 to provide experimental basis for follow-up experiments.③Cytotoxicity of RES to melanocytes: To observe the safeness of RES as a skin lightening and depigmenting agent, and find out its range of safe concentration to provide experimental basis for its clinical application.④The effects of RES on endothelin-1-induced melanogenesis and its inhibitory mechanism: To explore the efficacy of RES as a skin lightening and depigmenting agent, and a preliminary study the relevant mechanisms to provide experimental basis for its clinical applications. And the followings are our experimental methods and results.
Firstly, in the first experimental study, human melanocytes were obtained from 6 forekines from individuals aged from 6 to 11 years old. The average age is 8 years old. We cultured the cells in the keratinocyte serum free medium in the absence of TPA and CT. We used the characters that different time of adhesion and digestion between melanocytes and keratinocytes to remove keratinocytes. The fibroblasts were removed by two steps method. The cultured cells were identified with dopa staining and immunocytochemistry (anti-S100、HMB45 protein antibodies) methods. The results of them were positive. So we established a new method of culturing melanocytes in the absence of TPA and CT, and this method is stable and reliable. Secondly, in the second experimental study, human melanocytes were obtained from 6 forekines from individuals aged from 6 to 15 years old. The average age is 11 years old. Morphology was observed by invert microscope. The cell viability, tyrosinase activity, and melanin content were detected by CCK-8, DOPA oxidase and NaOH methods respectively. The results showed that endothelin-1 induced melanocyte dendricity, and stimulated cell viability at the concentration of 1~100 nM. It also increased tyrosinase activity and melanin content, with more marked effects at the concentration of 10 nM.
Thirdly, in the study of cytotoxicity of RES to melanocytes, human melanocytes were obtained from 6 forekines from individuals aged from 6 to 12 years old. The average age is 10 years old. We used the cck-8 method to detect the inhibition of cell proliferation and the Annexin V/PI double staining flow cytometric assay to detect the apoptosis rate of melanocyte. The results showed that RES could not inhibit cell growth and induce melanocyte apoptosis under the concentration of 10μM.
Finally, in the study of the effects of RES on endothelin-1- induced melanogenesis and its inhibitory mechanism, human melanocytes were obtained from 6 forekines from individuals aged from 6 to 15 years old. The average age is 12 years old. The melanin content, tyrosinase activity, tyrosinase and TRP-1 mRNA level were detected by NaOH, DOPA oxidase and semiquantitative RT-PCR methods, respectively. The results showed that RES could reduce endothelin-1-induced melanogenesis efficiently, and its mechanisms are mainly involved in the suppression of tyrosinase activity, and irrelevant to tyrosinase mRNA expression and TRP-1mRNA expression.
Our research results suggested that RES, acting as an endothelin antagonist, may inhibit tyrosinase activity to decrease melanin synthesis of melanocytes. Therefore, RES might be a skin whitening substance. The safe and effective concentration range is 0.1~10μM.
细胞因子与黑素细胞之间的关系已成为美白研究的另一重要方向。在皮肤,内皮素(endothelins, ET)表达于角质形成细胞,内皮素受体(endothelin receptor, EDNR)则表达于黑素细胞。已证实,ET可与黑素细胞膜上的内皮素受体相互作用并通过酪氨酸酶、酪氨酸酶相关蛋白-1(tyrosinase-related protein-1, TRP-1)、MAPK、PKC、PKA和酪氨酸激酶受体(receptor tyrosine kinase,KIT)这六条信号通路促进黑素细胞的增殖和黑素生成。因此,内皮素系统作为角质形成细胞和黑素细胞相互作用的桥梁,参与黑素细胞的增殖和黑素合成等过程,而内皮素拮抗剂可以通过拮抗ET的作用而抑制黑素细胞增殖及合成黑素。
已有报道证实白藜芦醇(resveratrol,RES)是一种非肽类内皮素拮抗剂。我们推测,RES作为一种内皮素拮抗剂可能从酪氨酸酶、TRP-1、MAPK、PKC、PKA和KIT这六条信号通路拮抗ET-1诱导的黑色素生成,从而达到美白祛斑的作用。
本课题主要目的是研究RES对ET-1介导的黑素细胞黑素合成的影响及其相关机制,并探讨其对黑素细胞的细胞毒性作用,为RES作为一种新型、安全和高效的皮肤美白祛斑物质提供一定的理论基础和实验依据。为了使该项研究顺利地完成,我们将整个实验细分为四个实验,分别是:①建立不含佛波醇酯(12-O-tetradecanoyl-phorbol-13-acetate,TPA)和霍乱毒素(cholera toxin,CT)体外培养正常人黑素细胞的方法:以避免TPA和CT改变黑素细胞的生物学特性,为后续的实验提供可靠的黑素细胞来源。②研究ET-1对培养的人黑素细胞生物学特性的影响:探讨ET-1对体外培养的正常人黑素细胞形态、增殖、酪氨酸酶活性及黑素合成的影响,并寻找一个最有效的调控浓度,为后续的实验提供实验基础。③RES对人黑素细胞的细胞毒性分析:以探讨RES应用于美白祛斑治疗的安全性,并寻找出其安全浓度范围,为其临床应用提供实验依据。④探讨RES对ET-1介导的黑素合成的影响及其相关机制:以探讨RES应用于美白祛斑治疗的有效性,并初步研究其相关机制,为其临床应用提供实验依据。针对以上四个实验目的,我们的实验方法和结果如下:
首先,在第一项实验研究中,我们取了青少年包皮环切术后包皮组织6例,年龄6-11岁,平均8岁,用不含TPA和CT的角质形成细胞无血清培养基(KC-SFM)培养、用差速贴壁法及胰酶差速消化法去除角质形成细胞、用两段法去除成纤维细胞,所培养出的细胞经多巴染色和抗S-100、抗HMB45蛋白抗体免疫细胞化学染色均呈阳性反应,证实为黑素细胞,从而建立了不含TPA和CT体外培养正常人黑素细胞的方法,该方法稳定、可靠。
其次,在第二项实验研究中,我们取了青少年包皮环切术后包皮组织6例,年龄6-15岁,平均11岁,分别用倒置显微镜观察、CCK-8法、多巴氧化法和NaOH法测定了ET-1对黑素细胞形态、增殖、酪氨酸酶活性和黑素含量的影响。该项实验结果提示:一定浓度的ET-1可有效地促进培养的正常人黑素细胞的增殖和树突形成,上调酪氨酸酶活性和增加黑素含量,10 nM的ET-1不仅能促进黑素细胞增殖及酪氨酸酶活性,而且此浓度下的黑素含量水平最高,因此10 nM是最有效的调控浓度。
再次,在分析RES对人黑素细胞的细胞毒性作用时,我们取了青少年包皮环切术后包皮组织3例,年龄6-12岁,平均10岁,用CCK-8法测定黑素细胞的增殖抑制率,Annexin V/PI双染色法流式细胞仪检测黑素细胞的凋亡率。该项实验结果显示,10μM浓度以下的RES对人黑素细胞无增殖抑制作用也无凋亡促进作用。
最后,在研究RES对ET-1介导的黑素合成的影响及其相关机制中,我们取了青少年包皮环切术后包皮组织9例,年龄6-15岁,平均12岁,采用NaOH法测定黑素含量,多巴氧化法测定黑素细胞的酪氨酸酶活性,半定量RT-PCR法测定酪氨酸酶mRNA和TRP-1 mRNA表达水平。我们的实验结果提示:0.1~10μM的RES可抑制ET-1介导的黑素合成,其主要机制可能是通过抑制酪氨酸酶活性起作用,而与酪氨酸酶mRNA和TRP-1mRNA的表达无关。
我们的实验结果表明,RES可作为一种内皮素拮抗剂,可以通过抑制酪氨酸酶活性减少黑素合成,且在10μM浓度以下时无细胞毒性作用。因此,RES可作为一种皮肤美白物质,其安全、有效的浓度范围是0.1~10μM。
In recent years, with the aging of population, environmental pollution, and global temperature rise, more and more people will develop pigmentation disorders, including age spots, melasma, chloasma, freckle, post inflammatory hyperpigmentation or sun-induced pigmented blemishes, etc. To discover the safer and more effective newer external-use skin lightening agents is now one of hot spots in the cosmetics research.
The relationship between cytokine and melanocyte has become another important research direction for the depigmenting agents. In epidermis, endothelins (ET) is expressed in keratinocytes and its receptor in melanocytes. It has been comfirmed that the ET can interact with its receptors located in melanocyte membrane to promote melanocyte proliferation and melanogenesis via six signal pathways including the tyrosinase, tyrosinase-related protein-1(TRP-1), MAPK, PKC, PKA and receptor tyrosine kinase(KIT) signal pathways. Thus, endothelins system, as a bridge connecting keratinocytes and melanocytes, participates in the proliferation and melanogenesis of melanocyte, and endothelin antagonist may inhibit cell proliferation and melanin synthesis of melanocytes through the antagonistic role of ET.
It has been comfirmed that resveratrol (RES) is a nonpeptide endothelin antagonist. So RES, acting as an endothelin antagonist, may efficiently antagonize the melanogenesis induced by ET-1 via six signal pathways including the tyrosinase, TRP-1, MAPK, PKC, PKA and KIT signal pathways.
The objective of this research project is to study the effects of RES on endothelin-1-induced melanogenesis and inhibitory mechanism, and explore cytotoxicity of RES on the normal cultured human melanocytes, to provide a theoretical basis and experimental evidenceand for development and application of RES as a novel, safe and highly effective skin lightening and depigmenting agent.The whole research project can be subdivided into the following four parts:①To establish a method of culturing melanocytes in the absence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and Cholera toxin(CT) : To avoid the oncogenicity of TPA and the change of biologic characteristics of cultured human melanocytes caused by adding TPA and CT, and provide the better melanocytes in biological characteristics for follow-up experiments.②To study effects of endothelin-1 on the biologic characteristics of cultured human melanocytes: To investigate the effects of endothelin-1 on the morphology, proliferation, tyrosinase activity and melanogenesis of normal human melanocytes cultured in vitro, and find one of the most effective regulatory concentration of ET-1 to provide experimental basis for follow-up experiments.③Cytotoxicity of RES to melanocytes: To observe the safeness of RES as a skin lightening and depigmenting agent, and find out its range of safe concentration to provide experimental basis for its clinical application.④The effects of RES on endothelin-1-induced melanogenesis and its inhibitory mechanism: To explore the efficacy of RES as a skin lightening and depigmenting agent, and a preliminary study the relevant mechanisms to provide experimental basis for its clinical applications. And the followings are our experimental methods and results.
Firstly, in the first experimental study, human melanocytes were obtained from 6 forekines from individuals aged from 6 to 11 years old. The average age is 8 years old. We cultured the cells in the keratinocyte serum free medium in the absence of TPA and CT. We used the characters that different time of adhesion and digestion between melanocytes and keratinocytes to remove keratinocytes. The fibroblasts were removed by two steps method. The cultured cells were identified with dopa staining and immunocytochemistry (anti-S100、HMB45 protein antibodies) methods. The results of them were positive. So we established a new method of culturing melanocytes in the absence of TPA and CT, and this method is stable and reliable. Secondly, in the second experimental study, human melanocytes were obtained from 6 forekines from individuals aged from 6 to 15 years old. The average age is 11 years old. Morphology was observed by invert microscope. The cell viability, tyrosinase activity, and melanin content were detected by CCK-8, DOPA oxidase and NaOH methods respectively. The results showed that endothelin-1 induced melanocyte dendricity, and stimulated cell viability at the concentration of 1~100 nM. It also increased tyrosinase activity and melanin content, with more marked effects at the concentration of 10 nM.
Thirdly, in the study of cytotoxicity of RES to melanocytes, human melanocytes were obtained from 6 forekines from individuals aged from 6 to 12 years old. The average age is 10 years old. We used the cck-8 method to detect the inhibition of cell proliferation and the Annexin V/PI double staining flow cytometric assay to detect the apoptosis rate of melanocyte. The results showed that RES could not inhibit cell growth and induce melanocyte apoptosis under the concentration of 10μM.
Finally, in the study of the effects of RES on endothelin-1- induced melanogenesis and its inhibitory mechanism, human melanocytes were obtained from 6 forekines from individuals aged from 6 to 15 years old. The average age is 12 years old. The melanin content, tyrosinase activity, tyrosinase and TRP-1 mRNA level were detected by NaOH, DOPA oxidase and semiquantitative RT-PCR methods, respectively. The results showed that RES could reduce endothelin-1-induced melanogenesis efficiently, and its mechanisms are mainly involved in the suppression of tyrosinase activity, and irrelevant to tyrosinase mRNA expression and TRP-1mRNA expression.
Our research results suggested that RES, acting as an endothelin antagonist, may inhibit tyrosinase activity to decrease melanin synthesis of melanocytes. Therefore, RES might be a skin whitening substance. The safe and effective concentration range is 0.1~10μM.
引文
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[13] Cho YH,Kim JH,Park SM,et al. New cosmetic agents for skin whitening from Angelica dahurica. J Cosmet Sci., 2006, 57(1):11-21.
[14] Kim DS,Park SH,Kwon SB, et al. Sphingosylphosphorylcholine-induced ERK activation inhibits melanin synthesis in human melanocytes. Pigment Cell Res., 2006, 19(2):146-153.
[15] Dong-Seok Kim,Yun-Mi Jeong,Ik-Kyu Park, et al. A new 2-Imino-1, 3-thiazoline derivative, KHG22394, inhibits melanin synthesis in mouse B16 melanoma cells. Biol. Pharm. Bull., 2007, 30(1):180-183.
[16] Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature,1988,332(6163): 411-5.
[17] Hara M, Yaar M, Gilchrest BA. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity. J Invest Dermatol, 1995,105 (6): 744-748.
[18]杨连春,王峰,刘敏,等.抗蛇毒中草药红背丝绸对内皮素-1的拮抗作用.中国新药杂志,2000,9(7):455-458.
[19] Yeon Mi Kim, Jieun Yun, Chong-Kil Lee, et al.Oxyresveratrol and hydroxystilbene compounds:inhibitory effect on tyrosinase and mechanism of action.the journal of biological chemistry, 2002,277(18): 16340-16344.
[20] Kawada N, Seki S, Inoue M, Kuroki T,et al. Effect of antioxidants, resveratrol, quercetin, and N-acetylcysteine, on the functions of cultured rat hepatic stellate cells and Kupffer cells. Hepatology, 1998, 27: 1265-1274.
[21] Louise E. Donnelly, Robert Newton, Gina E. Kennedy,et al. Anti-inflammatory effects of resveratrol in lung epithelial cells:molecular mechanisms Am J PhysiolLung Cell Mol Physiol,2004,287: 774-783.
[22] Yao Y, Tian T, Nan KJ. Research on resveratrol's mechanism of immunity in anti-aging. Zhong Yao Cai. , 2006, 29(5):464-467.
[23] Vohra BP, Planer W, Armon J, et al. Reduced endothelin converting enzyme-1 and endothelin-3 mRNA in the developing bowel of male mice may increase expressivity and penetrance of Hirschsprung disease-like distal intestinal aganglionosis. Dev Dyn, 2007, 236(1): 106-117.
[24] Ronit Lahav. Endothelin receptor B is required for the expansion ofmelanocyte precursors and malignant melanoma. Int. J. Dev. Biol, 2005, 49: 173-180.
[25] Imokawa G, Yada Y, Miyagishi M. Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes. J Biol Chem, 1992, 267(34): 24675-24680.
[26] Hachiya A, Kobayashi A, Yoshida Y, et al. Biphasic expression of two paracrine melanogenic cytokines, stem cell factor and endothelin-1, in ultraviolet B-induced human melanogenesis. Am J Pathol, 2004, 165(6): 2099-2109.
[27] Hachiya A, Kobayashi T, Takema Y, and Imokawa G. Biochemical characterization of endothelin-converting enzyme-1alpha in cultured skin-derived cells and its postulated role in the stimulation of melanogenesis in human epidermis. J Biol Chem, 2002, 277: 5395-5403.
[28] Adur J, Takizawa S, Uchide T, et al. High doses of ultraviolet-C irradiation increases vasoactive intestinal contractor/endothelin-2 expression in keratinocytes of the newborn mouse epidermis. Peptides, 2007 28(5): 1083-1094.
[29] Ma HJ, Zhu WY, Wang DG, et al. Endothelin-1 combined with extracellular matrix proteins promotes the adhesion and chemotaxis of amelanotic melanocytes from human hair follicles in vitro. Cell Biol Int, 2006, 30(12): 999-1006.
[30] Aoki H, Motohashi T, Yoshimura N, et al. Cooperative and indispensable roles of endothelin 3 and KIT signalings in melanocyte development. Dev Dyn, 2005, 233(2): 407-417.
[31] Hou L, Pavan WJ, Shin MK, et al. Cell-autonomous and cell non-autonomous signaling through endothelin receptor B during melanocyte development.Development, 2004, 131(14): 3239-3247.
[32] Sriwiriyanont P, Ohuchi A, Hachiya A, et al. Interaction between stem cell factor and endothelin-1: effects on melanogenesis in human skin xenografts. Lab Invest, 2006, 86(11): 1115-1125.
[33] Stanchina L, Baral V, Robert F, et al. Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development. Dev Biol, 2006, 295(1): 232-249.
[34] Yokoyama S, Takeda K, Shibahara S. SOX10, in combination with Sp1, regulates the endothelin receptor type B gene in human melanocyte lineage cells. FEBS J, 2006, 273(8): 1805-1820.
[35] Okazaki M, Yoshimura K, Uchida G, et al. Correlation between age and the secretions of melanocyte-stimulating cytokines in cultured keratinocytes and fibroblasts. Br J Dermatol, 2005,153 Suppl 2: 23-29.
[36]皮肖冰,曾凡钦,王晓霞,等.窄谱中波紫外线对寻常型白癜风患者皮肤组织液内皮素-1的影响.中国中西医结合皮肤性病学杂志,2007, 6(1): 11-13.
[37]陈菲菲,朱晓芳,王琴.白癜风患者血清中血管内皮素-1水平测定.黑龙江医学,2005, 29(11) : 851.
[38] Mangahas CR, dela Cruz GV, Schneider RJ, et al. Endothelin-1 upregulates MCAM in melanocytes. J Invest Dermatol, 2004, 123(6): 1135-1139.
[39] Bagnato A, Rosano L, Spinella F, et al. Endothelin B receptor blockade inhibits dynamics of cell interactions and communications in melanoma cell progression. Cancer Res, 2004, 64(4): 1436-1443.
[40] Mangahas CR, dela Cruz GV, Friedman-Jimenez G, et al. Endothelin-1 induces CXCL1 and CXCL8 secretion in human melanoma cells. J Invest Dermatol, 2005, 125(2): 307-311.
[41] Berger Y, Bernasconi CC, Juillerat-Jeanneret L. Targeting the endothelin axis in human melanoma: combination of endothelin receptor antagonism and alkylating agents. Exp Biol Med (Maywood), 2006, 231(6): 1111-1119.
[42] Spinella F, RosanòL, Di Castro V, et al. Endothelin-1 and endothelin-3 promote invasive behavior via hypoxia-inducible factor-1alpha in human melanoma cells.Cancer Res, 2007, 67(4): 1725-1734.
[43] Fang D, Leishear K, Nguyen TK, et al. Defining the conditions for the generation of melanocytes from human embryonic stem cells. Stem Cells, 2006, 24(7): 1668-1677.
[44] Eisenberg M,Lleweyn DM,Moran K,et al.Successful engraftment of cultured human epidermal allograft in a child with recessive dystrophic epidermolysis bullosa.Med J Aust 1987;147(10):520-521.
[45] Chen YF, Yang PY, Hung CM, et a1.Transplantation of autologous cultured Melanocytes for treatment of large segmental vitiligo.J Am Acad Dermatol 2001;44(3):543-545.
[46] Njoo MD, Westerhof W, Bos JD, et al. A systematic review of autologous transplantation Methosd in vitiligo.Arch Dermatol 1998;134:1543-1549.
[47]卢涛,刘源,金岩,等.两段法培养适合临床移植应用的黑素细胞的安全性研究.中国临床康复,2004;8(17):3242-3244.
[48] J. Picot.human cell culture protocols[M], second edition.Human press inc. Totowa, NJ 2006;13-31.
[49]丁国斌,陈璧,汤朝武.不同培养条件及时相对黑素细胞生物学特性的影响.中国临床康复,2002;6(24):3665-3666.
[50] Hirobe T,Furuya R,Ifuku O,et a1.Granulocyte-macrophage colony-stimulating factor is a keratinocyte-derived factor involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture.Exp Cell Res,2004;297(2):593-606.
[51] Horikawa T,Norris DA,Zekman T,et,al.Effective elimination of fibroblasts in cultures of melanocytes by lowering calcium concentration in TPA depleted medium following geneticin treatment.Pigment Cell Res,1996;9(2):58-62.
[52]屈颖,齐浩.黑素细胞的分离与培养综述.西安文理学院学报,2005;8(4):12-15.
[53]孙林潮,刘仲荣,赵小东,等.抗角蛋白自身抗体在黑素细胞无血清纯化培养中的应用.中国美容医学,2005;14(3):269-271.
[54]杨红,王克玉,张向红,等.过氧化氢对体外培养的黑素细胞形态及功能的影响.中国麻风皮肤病杂志,2006;22(7):538-540.
[55] Kadekaro AL,Kavanagh R Kanto H, et a1.alpha-Melanocortin and endothelin activate antiapoptotic pathways and reduce DNA damage in human melanocytes. Cancer Res, 2005, 65 (10): 4292-4299.
[56] Hara M, Yaar M, Gilchrest BA. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity. J Invest Dermatol, 1995, 105(6):744-748.
[57]杜娟,徐前喜,何培英,等.内皮缩血管肽1和促肾上腺皮质激素对体外培养的正常人黑素细胞增殖的影响.中华皮肤科杂志,2006,39(2):77-79.
[58] Monji A, Inoue H, Oshima H, et al. Tyrosinase induction and inactivation in normal cultured human melanocytes by endothelin-1.Int J Tissue React, 2005, 27(2):41-49.
[59] Imokawa G, Miyagishi M, Yada Y. Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis. J Invest Dermatol, 1995, 105(1):32-37.
[2] Andrzej Slominski, Desmond J.Tobin, Shigeki Shibahara, et al. Melanin pigmentation in mammalian skin and its hormonal regulation.Physiol Rev.,2004,84(4):1155-1228.
[3] Monji A,Inoue H,Oshima H,et al. Tyrosinase induction and inactivation in normal cultured human melanocytes by endothelin-1. Int J Tissue React.,2005,27 (2):41-49.
[4] Imokawa G,Yada Y, Kimura M, et al. Signalling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes. Biochem J., 1996,314 (Pt 1):305-312.
[5] Aoki H,Motohashi T,Yoshimura N,et al.Cooperative and indispensable roles of endothelin 3 and KIT signalings in melanocyte development. Dev Dyn., 2005, 233(2):407-417.
[6] Tada A, Suzuki I, Im S, et al. Endothelin-1 is a paracrine growth factor that modulates melanogenesis of human melanocytes and participates in their responses to ultraviolet radiation. Cell Growth Differ, 1998, 9(7):575-584.
[7] Yada V, Higuchi K, Imokawa G. Effects of endothelins on signal transduction and proliferation in human melanocytes. J Biol Chem, 1991, 266 (27):18352-18357.
[8] Cabanes J, Chazarra S, Garcia-Carmona F,et al. Kojic acid, a cosmetic skin whitening agent, is a slow-binding inhibitor of catecholase activity of tyrosinase J Pharm Pharmacol., 1994, 46(12): 982-985.
[9] Mineka Yoshimura, Yuko Watanabe, Kouichi Kasai, et al. Inhibitory effect of an ellagic acid-rich pomegranate extract on tyrosinase activity and ultraviolet-induced pigmentation. Biosci. Biotechnol. Biochem., 2005, 69 (12): 2368–2373.
[10] Rangkadilok N, Sitthimonchai S, Worasuttayangkurn L, et al. Evaluation of free radical scavenging and antityrosinase activities of standardized longan fruit extract. Food Chem Toxicol., 2007, 45(2):328-336.
[11] Lee KT, Lee KS, Jeong JH, et al. Inhibitory effects of Ramulus mori extracts on melanogenesis. J Cosmet Sci., 2003, 54(2):133-142.
[12] Ohguchi K, Banno Y, Akao Y, et al.Involvement of phospholipase D1 in melanogenesis of mouse B16 melanoma cells. J Biol Chem., 2004, 279(5):3408-3412.
[13] Cho YH,Kim JH,Park SM,et al. New cosmetic agents for skin whitening from Angelica dahurica. J Cosmet Sci., 2006, 57(1):11-21.
[14] Kim DS,Park SH,Kwon SB, et al. Sphingosylphosphorylcholine-induced ERK activation inhibits melanin synthesis in human melanocytes. Pigment Cell Res., 2006, 19(2):146-153.
[15] Dong-Seok Kim,Yun-Mi Jeong,Ik-Kyu Park, et al. A new 2-Imino-1, 3-thiazoline derivative, KHG22394, inhibits melanin synthesis in mouse B16 melanoma cells. Biol. Pharm. Bull., 2007, 30(1):180-183.
[16] Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature,1988,332(6163): 411-5.
[17] Hara M, Yaar M, Gilchrest BA. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity. J Invest Dermatol, 1995,105 (6): 744-748.
[18]杨连春,王峰,刘敏,等.抗蛇毒中草药红背丝绸对内皮素-1的拮抗作用.中国新药杂志,2000,9(7):455-458.
[19] Yeon Mi Kim, Jieun Yun, Chong-Kil Lee, et al.Oxyresveratrol and hydroxystilbene compounds:inhibitory effect on tyrosinase and mechanism of action.the journal of biological chemistry, 2002,277(18): 16340-16344.
[20] Kawada N, Seki S, Inoue M, Kuroki T,et al. Effect of antioxidants, resveratrol, quercetin, and N-acetylcysteine, on the functions of cultured rat hepatic stellate cells and Kupffer cells. Hepatology, 1998, 27: 1265-1274.
[21] Louise E. Donnelly, Robert Newton, Gina E. Kennedy,et al. Anti-inflammatory effects of resveratrol in lung epithelial cells:molecular mechanisms Am J PhysiolLung Cell Mol Physiol,2004,287: 774-783.
[22] Yao Y, Tian T, Nan KJ. Research on resveratrol's mechanism of immunity in anti-aging. Zhong Yao Cai. , 2006, 29(5):464-467.
[23] Vohra BP, Planer W, Armon J, et al. Reduced endothelin converting enzyme-1 and endothelin-3 mRNA in the developing bowel of male mice may increase expressivity and penetrance of Hirschsprung disease-like distal intestinal aganglionosis. Dev Dyn, 2007, 236(1): 106-117.
[24] Ronit Lahav. Endothelin receptor B is required for the expansion ofmelanocyte precursors and malignant melanoma. Int. J. Dev. Biol, 2005, 49: 173-180.
[25] Imokawa G, Yada Y, Miyagishi M. Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes. J Biol Chem, 1992, 267(34): 24675-24680.
[26] Hachiya A, Kobayashi A, Yoshida Y, et al. Biphasic expression of two paracrine melanogenic cytokines, stem cell factor and endothelin-1, in ultraviolet B-induced human melanogenesis. Am J Pathol, 2004, 165(6): 2099-2109.
[27] Hachiya A, Kobayashi T, Takema Y, and Imokawa G. Biochemical characterization of endothelin-converting enzyme-1alpha in cultured skin-derived cells and its postulated role in the stimulation of melanogenesis in human epidermis. J Biol Chem, 2002, 277: 5395-5403.
[28] Adur J, Takizawa S, Uchide T, et al. High doses of ultraviolet-C irradiation increases vasoactive intestinal contractor/endothelin-2 expression in keratinocytes of the newborn mouse epidermis. Peptides, 2007 28(5): 1083-1094.
[29] Ma HJ, Zhu WY, Wang DG, et al. Endothelin-1 combined with extracellular matrix proteins promotes the adhesion and chemotaxis of amelanotic melanocytes from human hair follicles in vitro. Cell Biol Int, 2006, 30(12): 999-1006.
[30] Aoki H, Motohashi T, Yoshimura N, et al. Cooperative and indispensable roles of endothelin 3 and KIT signalings in melanocyte development. Dev Dyn, 2005, 233(2): 407-417.
[31] Hou L, Pavan WJ, Shin MK, et al. Cell-autonomous and cell non-autonomous signaling through endothelin receptor B during melanocyte development.Development, 2004, 131(14): 3239-3247.
[32] Sriwiriyanont P, Ohuchi A, Hachiya A, et al. Interaction between stem cell factor and endothelin-1: effects on melanogenesis in human skin xenografts. Lab Invest, 2006, 86(11): 1115-1125.
[33] Stanchina L, Baral V, Robert F, et al. Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development. Dev Biol, 2006, 295(1): 232-249.
[34] Yokoyama S, Takeda K, Shibahara S. SOX10, in combination with Sp1, regulates the endothelin receptor type B gene in human melanocyte lineage cells. FEBS J, 2006, 273(8): 1805-1820.
[35] Okazaki M, Yoshimura K, Uchida G, et al. Correlation between age and the secretions of melanocyte-stimulating cytokines in cultured keratinocytes and fibroblasts. Br J Dermatol, 2005,153 Suppl 2: 23-29.
[36]皮肖冰,曾凡钦,王晓霞,等.窄谱中波紫外线对寻常型白癜风患者皮肤组织液内皮素-1的影响.中国中西医结合皮肤性病学杂志,2007, 6(1): 11-13.
[37]陈菲菲,朱晓芳,王琴.白癜风患者血清中血管内皮素-1水平测定.黑龙江医学,2005, 29(11) : 851.
[38] Mangahas CR, dela Cruz GV, Schneider RJ, et al. Endothelin-1 upregulates MCAM in melanocytes. J Invest Dermatol, 2004, 123(6): 1135-1139.
[39] Bagnato A, Rosano L, Spinella F, et al. Endothelin B receptor blockade inhibits dynamics of cell interactions and communications in melanoma cell progression. Cancer Res, 2004, 64(4): 1436-1443.
[40] Mangahas CR, dela Cruz GV, Friedman-Jimenez G, et al. Endothelin-1 induces CXCL1 and CXCL8 secretion in human melanoma cells. J Invest Dermatol, 2005, 125(2): 307-311.
[41] Berger Y, Bernasconi CC, Juillerat-Jeanneret L. Targeting the endothelin axis in human melanoma: combination of endothelin receptor antagonism and alkylating agents. Exp Biol Med (Maywood), 2006, 231(6): 1111-1119.
[42] Spinella F, RosanòL, Di Castro V, et al. Endothelin-1 and endothelin-3 promote invasive behavior via hypoxia-inducible factor-1alpha in human melanoma cells.Cancer Res, 2007, 67(4): 1725-1734.
[43] Fang D, Leishear K, Nguyen TK, et al. Defining the conditions for the generation of melanocytes from human embryonic stem cells. Stem Cells, 2006, 24(7): 1668-1677.
[44] Eisenberg M,Lleweyn DM,Moran K,et al.Successful engraftment of cultured human epidermal allograft in a child with recessive dystrophic epidermolysis bullosa.Med J Aust 1987;147(10):520-521.
[45] Chen YF, Yang PY, Hung CM, et a1.Transplantation of autologous cultured Melanocytes for treatment of large segmental vitiligo.J Am Acad Dermatol 2001;44(3):543-545.
[46] Njoo MD, Westerhof W, Bos JD, et al. A systematic review of autologous transplantation Methosd in vitiligo.Arch Dermatol 1998;134:1543-1549.
[47]卢涛,刘源,金岩,等.两段法培养适合临床移植应用的黑素细胞的安全性研究.中国临床康复,2004;8(17):3242-3244.
[48] J. Picot.human cell culture protocols[M], second edition.Human press inc. Totowa, NJ 2006;13-31.
[49]丁国斌,陈璧,汤朝武.不同培养条件及时相对黑素细胞生物学特性的影响.中国临床康复,2002;6(24):3665-3666.
[50] Hirobe T,Furuya R,Ifuku O,et a1.Granulocyte-macrophage colony-stimulating factor is a keratinocyte-derived factor involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture.Exp Cell Res,2004;297(2):593-606.
[51] Horikawa T,Norris DA,Zekman T,et,al.Effective elimination of fibroblasts in cultures of melanocytes by lowering calcium concentration in TPA depleted medium following geneticin treatment.Pigment Cell Res,1996;9(2):58-62.
[52]屈颖,齐浩.黑素细胞的分离与培养综述.西安文理学院学报,2005;8(4):12-15.
[53]孙林潮,刘仲荣,赵小东,等.抗角蛋白自身抗体在黑素细胞无血清纯化培养中的应用.中国美容医学,2005;14(3):269-271.
[54]杨红,王克玉,张向红,等.过氧化氢对体外培养的黑素细胞形态及功能的影响.中国麻风皮肤病杂志,2006;22(7):538-540.
[55] Kadekaro AL,Kavanagh R Kanto H, et a1.alpha-Melanocortin and endothelin activate antiapoptotic pathways and reduce DNA damage in human melanocytes. Cancer Res, 2005, 65 (10): 4292-4299.
[56] Hara M, Yaar M, Gilchrest BA. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity. J Invest Dermatol, 1995, 105(6):744-748.
[57]杜娟,徐前喜,何培英,等.内皮缩血管肽1和促肾上腺皮质激素对体外培养的正常人黑素细胞增殖的影响.中华皮肤科杂志,2006,39(2):77-79.
[58] Monji A, Inoue H, Oshima H, et al. Tyrosinase induction and inactivation in normal cultured human melanocytes by endothelin-1.Int J Tissue React, 2005, 27(2):41-49.
[59] Imokawa G, Miyagishi M, Yada Y. Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis. J Invest Dermatol, 1995, 105(1):32-37.