人表皮黑素细胞体外培养及过氧化氢对黑素细胞影响的实验研究
摘要
白癜风是一种常见的色素脱失性皮肤病，临床表现为局部或泛发性色素脱失斑。本病可发生于任何年龄，在我国人群中发病率约为0.1％-2.0％。白癜风多发于暴露部位，常给患者带来巨大的心理压力。白癜风的病因及发病机制尚未完全阐明，一般认为是具有遗传素质的个体，在内源性和外源性刺激的作用下导致的免疫、代谢、内分泌及神经精神功能的紊乱。目前病因主要有以下几个方面的假设：自身免疫学说；神经与精神化学学说；遗传学说；黑素细胞自身破坏学说和微量元素缺乏等学说。
近几年来随着自由基在医学上的深入研究，发现在白癜风患者的表皮内出现过氧化氢的过量聚集，过氧化氢是一种较强的氧化剂，它可以与金属离子或氧离子发生反应，产生羟自由基。羟自由基可以引起脂质过氧化反应，损伤细胞。表皮的氧化应激状态可能降低了某些酶的活性，造成黑素细胞的损伤，导致色素脱失。
本试验以过氧化氢作为外源性氧化剂，通过向体外培养的黑素细胞中加入过氧化氢，使黑素细胞处于氧化应激的状态，观察细胞在形态超微结构及功能上的改变，探讨氧化应激与白癜风发病机制的关系。本研究旨在探讨氧化应激对黑素细胞的损伤及其与白癜风发病的关系，为白癜风的预防和治疗提供实验依据。
第一部分 黑素细胞的体外培养及生物学鉴定
目的 为应用培养的黑素细胞研究白癜风的发病机制，对正常人表皮的黑素细胞进行体外原代及传代培养，并对黑素细胞的生物学特性进行鉴定。
方法 采用MCDB254培养基对正常人表皮黑素细胞进行体外培养，采用HMB-45单克隆抗体免疫组化法对培养细胞进行鉴定，透射电镜观察细胞的超微结构，确定培养细胞为黑素细胞且具有正常的生物学特征
Vitiligo is a depigmenting disease of the skin ,characterized by destruction of MC in lesions of vitiligo , resulting in patches of depigmentation. In China, the incidence of vitiligo in patients is about 1% to 2%. It can condition the behavior of people suffering from vitiligo and can deeply affect their personal relationships. The etiology is still unknown and pathogenesis has not been completely clarified so far, various hypotheses have been proposed, they include the genetic hypotheses, the autoimmune hypotheses , the autocytotoxic hypotheses, the neural hypotheses, and the lack of microelement hypotheses.
In recent years , Increase levels of H_2O_2 were demonstrated in the epidermis of both lesional and nonlesional skin of vitiligo patients, H_2O_2 levels increase in response to oxidative stress. An imbalance of antioxidants was found in the epidermis, correlated to an increased reactive oxygen species(ROS) production. ROS can interact with macrom olecules such as proteins, membrance lipids and nucleic acids, and fina lly cause cells damaged.
In this study , Normal melanocytes were cultured with H_2O_2 in different dosages(0-0. 5mmol/L) and different time(0—60minute), the appearance and function of the cells were analysed .The aim of this study was to evaluate the effect of H_2O_2 on the melanocyte in vitro cultured and the relationship between oxidative stress and vitiligo. Part One Culture and biological characteristics of melanocytes Objective To indentify the biological characteristics of MC cultured in
近几年来随着自由基在医学上的深入研究，发现在白癜风患者的表皮内出现过氧化氢的过量聚集，过氧化氢是一种较强的氧化剂，它可以与金属离子或氧离子发生反应，产生羟自由基。羟自由基可以引起脂质过氧化反应，损伤细胞。表皮的氧化应激状态可能降低了某些酶的活性，造成黑素细胞的损伤，导致色素脱失。
本试验以过氧化氢作为外源性氧化剂，通过向体外培养的黑素细胞中加入过氧化氢，使黑素细胞处于氧化应激的状态，观察细胞在形态超微结构及功能上的改变，探讨氧化应激与白癜风发病机制的关系。本研究旨在探讨氧化应激对黑素细胞的损伤及其与白癜风发病的关系，为白癜风的预防和治疗提供实验依据。
第一部分 黑素细胞的体外培养及生物学鉴定
目的 为应用培养的黑素细胞研究白癜风的发病机制，对正常人表皮的黑素细胞进行体外原代及传代培养，并对黑素细胞的生物学特性进行鉴定。
方法 采用MCDB254培养基对正常人表皮黑素细胞进行体外培养，采用HMB-45单克隆抗体免疫组化法对培养细胞进行鉴定，透射电镜观察细胞的超微结构，确定培养细胞为黑素细胞且具有正常的生物学特征
Vitiligo is a depigmenting disease of the skin ,characterized by destruction of MC in lesions of vitiligo , resulting in patches of depigmentation. In China, the incidence of vitiligo in patients is about 1% to 2%. It can condition the behavior of people suffering from vitiligo and can deeply affect their personal relationships. The etiology is still unknown and pathogenesis has not been completely clarified so far, various hypotheses have been proposed, they include the genetic hypotheses, the autoimmune hypotheses , the autocytotoxic hypotheses, the neural hypotheses, and the lack of microelement hypotheses.
In recent years , Increase levels of H_2O_2 were demonstrated in the epidermis of both lesional and nonlesional skin of vitiligo patients, H_2O_2 levels increase in response to oxidative stress. An imbalance of antioxidants was found in the epidermis, correlated to an increased reactive oxygen species(ROS) production. ROS can interact with macrom olecules such as proteins, membrance lipids and nucleic acids, and fina lly cause cells damaged.
In this study , Normal melanocytes were cultured with H_2O_2 in different dosages(0-0. 5mmol/L) and different time(0—60minute), the appearance and function of the cells were analysed .The aim of this study was to evaluate the effect of H_2O_2 on the melanocyte in vitro cultured and the relationship between oxidative stress and vitiligo. Part One Culture and biological characteristics of melanocytes Objective To indentify the biological characteristics of MC cultured in
引文
1 朱铁君．色素性皮肤病．北京医科大学中国协和医科大学联合出版社．1996年
2 Eisinger M, Marko O. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc Nail Acad Sci USA, 1982, 79(6): 2018-22
3 赵辩，黄秋玲，毕志刚等．人正常黑素细胞体外纯培养及其细胞生物学鉴定．临床皮肤科杂志，1991，20：226
4 Kim HI, Kim NS, Park YK. Biologic characteristics of cultured human vitiligo melanocytes. Int J Dermatol 1994, 33(8): 556-62
5 Abdel Malek Z, Swope VB, Pallas J, et al. Mitogenic melanogenic and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens. J Cellular Physiology, 1992, 150(2): 416-25
6 Barrett AW, Raja AM. The immunohistochemical identification of human oral mucosal melanocytes. Arch Oral Biol, 1997, 42(1): 77-81
7 Eves PC, Beck AJ, Shard AG, et al. A chemically defined surface for the co-culture of melanocytes and keratinocytes. Biomaterials, 2005, 26(34): 7068-7081
8 杨吉成，单礼华，周建华等．医用细胞工程．第二版．上海交通大学出版社．2003年
9 Lin SJ, Jee SH, Hsaio WC, et al. Formation of melanocyte spheroids on the chitosan-coated surface. Biomaterials, 2005, 26(12): 1413-1422
10 宋今丹，章敬波，张世馥等．组织和细胞培养技术．北京：人民卫生出版社．2002年
11 Boissy RE, Nordlund JJ. Molecular basis of congenital hypopigmentary disorders in humans: a review. Pigment Cell Res, 1997, 10 (1-2): 12-24
12 mengeaud V, Ortonne JP. Regulation of melanogenesis induced by 5-o-methoxypsoral without ultraviolet light in murine melanoma cells. Pigment Cell Res, 1994, 7: 245-54
13 Goding CR, Fisher DE. Regulation of melanocyte differentiation and growth. Cell Growth Differ, 1997, 8 (9) :935-940
14 De Leeuw SM, Smit NP, Van Veldhoven M, et al. Melanin content of cultured human melanocytes And UV-induced cytotoxicity.J Photochemistry and Photobiogy B, 2001, 61(3):106-113
15 Halaban R. The regulation of Normal melanocyte proliferation. Pigment Cell Res, 2000,13(1): 4-14
16 Eisinger M, Marko 0. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc Natl Acad Sci USA, 1982, 79(6):2018-22
17 Horikawa T, Norris DA, Zekman T, et al. Efective 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
18 Mahalingam H, Vaughn J, Novotny J, et al. Regulation of melanogenesis in B16 mouse melanoma cells by protein kinase C. J Cell Physiol, 1996, 168(3):549-58
19 AritaY, Driscoll KR, Weinstein IB. Growth of human melanocyte cultures supported by 12-0-tetradecanoylphorbol-13-acetate is mediated through protein kinase C activation. Cancer Res, 1992,15;52(16):4514-4521
20 Njoo MD, Westerhof W, Bos JD, et al. A systematic review of autologous transplantation Methods in vitiligo. Arch Dermatol, 1998,134(12): 1543-1549
21 Virador VM, Kobayashi N, Matsunage J, et al. A standardized protocol for assessing regulators of pigmentation. Anal Biochem, 1999,1;270(2): 207-19
22 Yaar M, Gilchrest BA. Human melanocyte growth and differentiation: a decade of new data. J Invest Dermato, 1991, 97(4):611-617
23 Halaban R. Signal transduction in normal and malignant melanocytes. Pigment Cell Res, 1994, 7(2):89-95
24 Kim NS, Cho JH, Kang WH. Behavioral differences between donor site-matched adult and neonatal melanocytes in culture. Arch Dermatol Res, 2000,292(5):233-239
25 Gilchrest BA, Vrabel MA, Flynn E, et al. selective cultivation of human melanocytes from newborn and adult epidermal. J Invest Dermatol, 1984, 83(5): 370-376
26 Le Poole IC, van den Wijngaard RM, Westerhof W, et al. Presence or absence of melanocytes in vitiligo lesions: an immunohistochemical investigation. J Invest Dermatol, 1993,100(6):816-822
27 Adema GJ, de Boer AJ, Vogel AM, et al. Molecular characterization of the melanocyte lineage-specific antigen gp100. J Biol Chem, 1994, 269 (31):20126-33
28 Pianigiani E, Andreassi A, Andreassi L. Autografts and cultured epidermis in the treatment of vitiligo. Clinics in Dermatology, 2005 23(4): 424-429
29 Giovannelli L, Bellandi S, Pitozzi V, et al. Increased oxidative DNA damage in mononuclear leukocytes in vitiligo. Mutation Research , 2004, 556 (1-2) : 101-106
30 Cucchi ML, Frattini P, Santagostino G. Higher plasma catecholamine and metabolite levels in the early phase of non-segmental vitiligo. Pigment Cell Res, 2000,13:28-32
31 Huang CL, Nordlund JJ, Boissy R. Vitiligo: a manifestation of apoptosis? Am J Clin Dermatol, 2002, 3(5):301-8
32 Schallreuter KU, Wood JM, Pittelkow MR, et al. Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin . Science, 1994 263(5152): 1444-1446
33 Maresca V, Roccella M, Roccella F, et al. Increased sensitivity to peroxidative agents as a possible pathogenic factor of melanocyte damage in vitiligo, J Invest Dermatol, 1997, 109(3): 1081-1085.
34 Schallreuter KU, Moore J, Wood J M, et al. Epidermal H_2O_2 accumulation alter tetrahydrobiopterin (6BH4) recycling in vitiligo: Identifi-cation of a general mechanism in regulation of all 6BH4 dependent processes. J Invest Oermatol, 2001, 116(1): 167-174.
35 Pelle E, Mammone T, Maes DE, et al. Keratinocytes act as a source of reactive oxygen species by transferring hydrogen peroxide to melanocytes. J Invest Dermatol, 2005, 124(4): 793-797
36 杨壮群，王正辉，虎小毅等．α-硫辛酸对体外培养的黑素细胞影响的实验研究．中国皮肤性病学杂志，2004，18(2)：71-73
37 Sarangarajan R, Shumaker H, Soleimani M, et al. Molecular and functional characterization of sodium hydrogen exchanger in skin as well as cultured keratinocytes and melanocytes. Biochim Biophys Acta, 2001, 1511(1): 181-192
38 Boisseau-Garsaud AM, Garsaud P, Cales-Quist D, et al. Epidemiology of vitiligo in the French West Indies (Isle ofgartinique). Int J Dermatol, 2000, 39(1): 18-20.
39 De Leeuw SM, Smit NP, Van Veldhoven M, et al. Melanin content of cultured human melanocytes and UV-induced cytotoxicity. J Photochem Photobiol B, 2001, 30;61(3): 106-113
40 Hoogduijn MJ, Cemeli E, Ross K, et al. Melanin protects melanocytes and keratinocytes against H202-induced DNA strand breaks through its ability to bind Ca2+. Experimental Cell Research, 2004, 10: 294(1): 60-67
41 Im S, Hann SK, Kim HI, et al. Biologic characteristics of cultured human vitiligo melanocytes. Int J Dermatol, 1994, 33(8): 556-62
42 Agrawal D, Shajil EM, Marfatia YS, et al. Study on the antioxidant status of vitiligo patients of different age groups in Baroda. Pigment Cell Res, 2004 17(3): 289-294
43 Boissy RE, Liu YY, Medrano EE, et al. Structural aberration of the rough endoplasmic reticulum and melanosome compartmentalization in long-term cultures of melanocytes from vitiligo patients. J Invest Dermatol, 1991, 97(3): 395-404
44 张汝芝，朱文元．白癜风的病因及儿童白癜风．临床皮肤科杂志，2002，10(31)：669-672
45 朱光斗，周萍英．白癜风与染色体畸变．临床皮肤科杂志，1995，24(4)：223-225
46 刘庆梅，王朝凤．白癜风患者血清，皮损铜锌元素及血清自由基清除系统水平检测．临床皮肤科杂志，1997，26(3)：171-173
47 Rokos H, Beazley WD, Schallreuter KU. Oxidative stress in vutiligo: photo-oxidation of pterins produces H_2O_2 and pterin-6-carboxylic acid. Biochem Biophys Res Commun, 2002, 12: 292 (4): 805-811
48 Jimenez-Cervantes C, Martinez-Esparza M, Perez C, et al. Inhibition of melanogenesis in response to oxidative stress: transient downregulation of melanocyte differentiation markers and possible involvement of microphthalmia transcription factor. J cell science, 2001, 114(12): 2335-2344
2 Eisinger M, Marko O. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc Nail Acad Sci USA, 1982, 79(6): 2018-22
3 赵辩，黄秋玲，毕志刚等．人正常黑素细胞体外纯培养及其细胞生物学鉴定．临床皮肤科杂志，1991，20：226
4 Kim HI, Kim NS, Park YK. Biologic characteristics of cultured human vitiligo melanocytes. Int J Dermatol 1994, 33(8): 556-62
5 Abdel Malek Z, Swope VB, Pallas J, et al. Mitogenic melanogenic and cAMP responses of cultured neonatal human melanocytes to commonly used mitogens. J Cellular Physiology, 1992, 150(2): 416-25
6 Barrett AW, Raja AM. The immunohistochemical identification of human oral mucosal melanocytes. Arch Oral Biol, 1997, 42(1): 77-81
7 Eves PC, Beck AJ, Shard AG, et al. A chemically defined surface for the co-culture of melanocytes and keratinocytes. Biomaterials, 2005, 26(34): 7068-7081
8 杨吉成，单礼华，周建华等．医用细胞工程．第二版．上海交通大学出版社．2003年
9 Lin SJ, Jee SH, Hsaio WC, et al. Formation of melanocyte spheroids on the chitosan-coated surface. Biomaterials, 2005, 26(12): 1413-1422
10 宋今丹，章敬波，张世馥等．组织和细胞培养技术．北京：人民卫生出版社．2002年
11 Boissy RE, Nordlund JJ. Molecular basis of congenital hypopigmentary disorders in humans: a review. Pigment Cell Res, 1997, 10 (1-2): 12-24
12 mengeaud V, Ortonne JP. Regulation of melanogenesis induced by 5-o-methoxypsoral without ultraviolet light in murine melanoma cells. Pigment Cell Res, 1994, 7: 245-54
13 Goding CR, Fisher DE. Regulation of melanocyte differentiation and growth. Cell Growth Differ, 1997, 8 (9) :935-940
14 De Leeuw SM, Smit NP, Van Veldhoven M, et al. Melanin content of cultured human melanocytes And UV-induced cytotoxicity.J Photochemistry and Photobiogy B, 2001, 61(3):106-113
15 Halaban R. The regulation of Normal melanocyte proliferation. Pigment Cell Res, 2000,13(1): 4-14
16 Eisinger M, Marko 0. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc Natl Acad Sci USA, 1982, 79(6):2018-22
17 Horikawa T, Norris DA, Zekman T, et al. Efective 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
18 Mahalingam H, Vaughn J, Novotny J, et al. Regulation of melanogenesis in B16 mouse melanoma cells by protein kinase C. J Cell Physiol, 1996, 168(3):549-58
19 AritaY, Driscoll KR, Weinstein IB. Growth of human melanocyte cultures supported by 12-0-tetradecanoylphorbol-13-acetate is mediated through protein kinase C activation. Cancer Res, 1992,15;52(16):4514-4521
20 Njoo MD, Westerhof W, Bos JD, et al. A systematic review of autologous transplantation Methods in vitiligo. Arch Dermatol, 1998,134(12): 1543-1549
21 Virador VM, Kobayashi N, Matsunage J, et al. A standardized protocol for assessing regulators of pigmentation. Anal Biochem, 1999,1;270(2): 207-19
22 Yaar M, Gilchrest BA. Human melanocyte growth and differentiation: a decade of new data. J Invest Dermato, 1991, 97(4):611-617
23 Halaban R. Signal transduction in normal and malignant melanocytes. Pigment Cell Res, 1994, 7(2):89-95
24 Kim NS, Cho JH, Kang WH. Behavioral differences between donor site-matched adult and neonatal melanocytes in culture. Arch Dermatol Res, 2000,292(5):233-239
25 Gilchrest BA, Vrabel MA, Flynn E, et al. selective cultivation of human melanocytes from newborn and adult epidermal. J Invest Dermatol, 1984, 83(5): 370-376
26 Le Poole IC, van den Wijngaard RM, Westerhof W, et al. Presence or absence of melanocytes in vitiligo lesions: an immunohistochemical investigation. J Invest Dermatol, 1993,100(6):816-822
27 Adema GJ, de Boer AJ, Vogel AM, et al. Molecular characterization of the melanocyte lineage-specific antigen gp100. J Biol Chem, 1994, 269 (31):20126-33
28 Pianigiani E, Andreassi A, Andreassi L. Autografts and cultured epidermis in the treatment of vitiligo. Clinics in Dermatology, 2005 23(4): 424-429
29 Giovannelli L, Bellandi S, Pitozzi V, et al. Increased oxidative DNA damage in mononuclear leukocytes in vitiligo. Mutation Research , 2004, 556 (1-2) : 101-106
30 Cucchi ML, Frattini P, Santagostino G. Higher plasma catecholamine and metabolite levels in the early phase of non-segmental vitiligo. Pigment Cell Res, 2000,13:28-32
31 Huang CL, Nordlund JJ, Boissy R. Vitiligo: a manifestation of apoptosis? Am J Clin Dermatol, 2002, 3(5):301-8
32 Schallreuter KU, Wood JM, Pittelkow MR, et al. Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin . Science, 1994 263(5152): 1444-1446
33 Maresca V, Roccella M, Roccella F, et al. Increased sensitivity to peroxidative agents as a possible pathogenic factor of melanocyte damage in vitiligo, J Invest Dermatol, 1997, 109(3): 1081-1085.
34 Schallreuter KU, Moore J, Wood J M, et al. Epidermal H_2O_2 accumulation alter tetrahydrobiopterin (6BH4) recycling in vitiligo: Identifi-cation of a general mechanism in regulation of all 6BH4 dependent processes. J Invest Oermatol, 2001, 116(1): 167-174.
35 Pelle E, Mammone T, Maes DE, et al. Keratinocytes act as a source of reactive oxygen species by transferring hydrogen peroxide to melanocytes. J Invest Dermatol, 2005, 124(4): 793-797
36 杨壮群，王正辉，虎小毅等．α-硫辛酸对体外培养的黑素细胞影响的实验研究．中国皮肤性病学杂志，2004，18(2)：71-73
37 Sarangarajan R, Shumaker H, Soleimani M, et al. Molecular and functional characterization of sodium hydrogen exchanger in skin as well as cultured keratinocytes and melanocytes. Biochim Biophys Acta, 2001, 1511(1): 181-192
38 Boisseau-Garsaud AM, Garsaud P, Cales-Quist D, et al. Epidemiology of vitiligo in the French West Indies (Isle ofgartinique). Int J Dermatol, 2000, 39(1): 18-20.
39 De Leeuw SM, Smit NP, Van Veldhoven M, et al. Melanin content of cultured human melanocytes and UV-induced cytotoxicity. J Photochem Photobiol B, 2001, 30;61(3): 106-113
40 Hoogduijn MJ, Cemeli E, Ross K, et al. Melanin protects melanocytes and keratinocytes against H202-induced DNA strand breaks through its ability to bind Ca2+. Experimental Cell Research, 2004, 10: 294(1): 60-67
41 Im S, Hann SK, Kim HI, et al. Biologic characteristics of cultured human vitiligo melanocytes. Int J Dermatol, 1994, 33(8): 556-62
42 Agrawal D, Shajil EM, Marfatia YS, et al. Study on the antioxidant status of vitiligo patients of different age groups in Baroda. Pigment Cell Res, 2004 17(3): 289-294
43 Boissy RE, Liu YY, Medrano EE, et al. Structural aberration of the rough endoplasmic reticulum and melanosome compartmentalization in long-term cultures of melanocytes from vitiligo patients. J Invest Dermatol, 1991, 97(3): 395-404
44 张汝芝，朱文元．白癜风的病因及儿童白癜风．临床皮肤科杂志，2002，10(31)：669-672
45 朱光斗，周萍英．白癜风与染色体畸变．临床皮肤科杂志，1995，24(4)：223-225
46 刘庆梅，王朝凤．白癜风患者血清，皮损铜锌元素及血清自由基清除系统水平检测．临床皮肤科杂志，1997，26(3)：171-173
47 Rokos H, Beazley WD, Schallreuter KU. Oxidative stress in vutiligo: photo-oxidation of pterins produces H_2O_2 and pterin-6-carboxylic acid. Biochem Biophys Res Commun, 2002, 12: 292 (4): 805-811
48 Jimenez-Cervantes C, Martinez-Esparza M, Perez C, et al. Inhibition of melanogenesis in response to oxidative stress: transient downregulation of melanocyte differentiation markers and possible involvement of microphthalmia transcription factor. J cell science, 2001, 114(12): 2335-2344