维甲酸对小鼠B16黑素瘤细胞和正常人黑素细胞的影响
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摘要
黑素细胞是合成和分泌黑素的树枝状细胞，位于表皮和真皮交界处，每个黑素细胞与其周围约36个角质形成细胞在结构和功能上存在着密切的联系，形成了表皮黑素单位。黑素细胞几乎遍布所有组织，最常见于表皮、毛囊等处。黑素分为优黑素和褐黑素两种，其生物合成是一个由酪氨酸酶催化体内酪氨酸羟化而启动的一系列生化反应过程。酪氨酸酶是黑素生成的限速酶，其数量和活性决定着黑素生成的速度和产量。因为黑素是决定皮肤颜色的主要色素，关于黑素细胞及黑素生物合成调控问题的研究已成为近几年来科研的热点之一。人们在体外培养黑素细胞的基础上，对黑素细胞与周围细胞之间的调节、黑素细胞与内分泌和免疫系统的关系、细胞因子及炎性介质的作用、以及酪氨酸酶基因家族表达的调控等方面进行了很多的研究。对于临床医疗工作具有实际指导意义的是研究某些安全有效的药物对黑素细胞的功能、尤其是黑素合成的调节作用。维甲酸是目前逐渐得到广泛应用的一类药物，它具有多种药理活性。在临床实践中，人们发现维甲酸对人的肤色有影响，但缺乏确实的临床与实验证明。
本实验基于体外培养小鼠B16黑素瘤细胞和正常人黑素细胞，对不同浓度的维甲酸及其与氢醌协同对黑素细胞的作用进行研究，并通过12-O-十四烷酰佛波醇-13-醋酸酯(TPA)、异丁基甲基黄嘌呤(IBMX)和霍乱毒素(CT)对维甲酸诱导小鼠B16黑素瘤细胞的影响，对其调控机制进行了初步探讨。将来自同一原瓶的小鼠B16黑素瘤细胞或正常人黑素细胞转种至96孔板，调节细胞初始接种浓度为5*10~5个／ml左右。小鼠B16黑素瘤细胞接种24小时，正常人黑素细胞接种48小时后，用添加维甲酸及其它药物的新鲜培养基换液一次。继续孵育72小时后检测酪氨酸酶活性、黑素含量和黑素细胞增殖率。
实验结果显示：维甲酸在较高浓度(＞500μmol／L)可以下调酪氨酸酶活性和黑素含量，对细胞无明显毒性作用，同时还有促进黑素细胞增殖的作用。较高浓度的维甲酸(＞500μmol／L)与氢醌可以互相协同，增强彼此下调酪氨酸
医学系2000级硕士研究生学位论文
酶活性和黑素含量的作用，而且维甲酸还可以减轻氢酮对细胞增殖的抑制作
用，达到了保护细胞免受伤害的效果，使作用更加安全有效。若将维甲酸以
适当浓度与氢醒混合外用，对于色素沉着性疾病可能会有较好疗效，但尚有
待临床研究的进一步证实。维甲酸对TPA、IBMX、CT提高小鼠B16黑素瘤细
胞酪氨酸酶活性和黑素含量的作用有轻微的抑制，在1000 p Inol/L以上时与
IB袱和CT的协同促增殖作用较强。虽然我们做的是细胞和蛋白水平的研究，
但不同于氢酮的作用机制，维甲酸很可能是通过基因水平的调节而发挥作用
的。维甲酸很可能是与核受体(RAR)结合后通过以下三条信号转导途径调节
黑素细胞增殖和黑素生成，即二酷酞甘油/蛋白激酶C(DAG/PKC)途径、环
磷酸腺昔/蛋白激酶A(c AMP/PKA)途径和丝裂原激活的蛋白激酶(做PK)级
联途径。而维甲酸对黑素细胞的具体基因调控机制还有待于进一步的研究。
The melanocyte is the pigment-producing dendritic cell of the epidermis. Melanocytes reside in the basal layer in normal adult epidermis. One melanocyte is therefore in contact with about 36 keratinocytes; together they form the so-called epidermal melanin unit. Melanocytes are most commonly in epidermis and follicle. Melanin involves eumelanin and pheomelanin, if s synthesized through a series of stages in which the enzyme tyrosinase acts on melanin precursors to produce the densely pigmented granules. Tyrosinase is the key enzyme of melanogenesis and determine the synthesis and quantity of melanin. Because melanin is the main pigment that determines the skin color, some researchers pay much attention to the modulation of melanocytes and melagenesis. The relationship between melanocytes and other cells, the contact between melanocytes and endocrine system or immune system, the effect of cytokine and the gene family of tyronase were investigated based on the culture of melanocytes in vitro. The most practical study is to find some drugs which are safe and effective to regulate the melanocytes and melagenesis. Retinoids are used abroad now and with much pharmacological activity. The effect of it on skin color is found in clinic.
We investigated the effects of retinoic acid and other drugs on B16 murine melanoma cells and normal human melanocytes in vitro, in search of the most effective concentration and get insight on the mechanisms involved. B16 murine melanoma cells and normal human melanocytes were cultured in vitro. The cells from the same original bottle were transplanted to the 96 well cell culture cluster. The cell concentration were about 5*105/ml. B16 murine melanoma cells were incubated for 24 hours, while the normal human melanocytes were incubated for 48 hours. Then the culture medium was replaced and retinoic acid or other drugs
were added to it. After treating for 72 hours, tyrosinase activity, melanin content and cell proliferation rate were detected.
The results showed that retinoic acid exhibited an inhibitory effect on tyrosinase activity and melanin production at high concentration (>500 μmol/L), and could promote the cell proliferation. Retinoic acid and hydroquinone could be cooperative at high concentration(>500μmol/L), enhanced the down regulation of tyrosinase activity and melanin content. Retinoic acid could also mitigate the inhibitory effect of hydroquinone on cell proliferation, so as to protect the cells from injure. Retinoic acid showed slight inhibitory effect on the up regulation of tyrosinase activity and melanin content which were induced by TPA,IBMX and CT,it could promote the cell proliferation with IBMX and CT over 1000 μ mol/L.
本实验基于体外培养小鼠B16黑素瘤细胞和正常人黑素细胞，对不同浓度的维甲酸及其与氢醌协同对黑素细胞的作用进行研究，并通过12-O-十四烷酰佛波醇-13-醋酸酯(TPA)、异丁基甲基黄嘌呤(IBMX)和霍乱毒素(CT)对维甲酸诱导小鼠B16黑素瘤细胞的影响，对其调控机制进行了初步探讨。将来自同一原瓶的小鼠B16黑素瘤细胞或正常人黑素细胞转种至96孔板，调节细胞初始接种浓度为5*10~5个／ml左右。小鼠B16黑素瘤细胞接种24小时，正常人黑素细胞接种48小时后，用添加维甲酸及其它药物的新鲜培养基换液一次。继续孵育72小时后检测酪氨酸酶活性、黑素含量和黑素细胞增殖率。
实验结果显示：维甲酸在较高浓度(＞500μmol／L)可以下调酪氨酸酶活性和黑素含量，对细胞无明显毒性作用，同时还有促进黑素细胞增殖的作用。较高浓度的维甲酸(＞500μmol／L)与氢醌可以互相协同，增强彼此下调酪氨酸
医学系2000级硕士研究生学位论文
酶活性和黑素含量的作用，而且维甲酸还可以减轻氢酮对细胞增殖的抑制作
用，达到了保护细胞免受伤害的效果，使作用更加安全有效。若将维甲酸以
适当浓度与氢醒混合外用，对于色素沉着性疾病可能会有较好疗效，但尚有
待临床研究的进一步证实。维甲酸对TPA、IBMX、CT提高小鼠B16黑素瘤细
胞酪氨酸酶活性和黑素含量的作用有轻微的抑制，在1000 p Inol/L以上时与
IB袱和CT的协同促增殖作用较强。虽然我们做的是细胞和蛋白水平的研究，
但不同于氢酮的作用机制，维甲酸很可能是通过基因水平的调节而发挥作用
的。维甲酸很可能是与核受体(RAR)结合后通过以下三条信号转导途径调节
黑素细胞增殖和黑素生成，即二酷酞甘油/蛋白激酶C(DAG/PKC)途径、环
磷酸腺昔/蛋白激酶A(c AMP/PKA)途径和丝裂原激活的蛋白激酶(做PK)级
联途径。而维甲酸对黑素细胞的具体基因调控机制还有待于进一步的研究。
The melanocyte is the pigment-producing dendritic cell of the epidermis. Melanocytes reside in the basal layer in normal adult epidermis. One melanocyte is therefore in contact with about 36 keratinocytes; together they form the so-called epidermal melanin unit. Melanocytes are most commonly in epidermis and follicle. Melanin involves eumelanin and pheomelanin, if s synthesized through a series of stages in which the enzyme tyrosinase acts on melanin precursors to produce the densely pigmented granules. Tyrosinase is the key enzyme of melanogenesis and determine the synthesis and quantity of melanin. Because melanin is the main pigment that determines the skin color, some researchers pay much attention to the modulation of melanocytes and melagenesis. The relationship between melanocytes and other cells, the contact between melanocytes and endocrine system or immune system, the effect of cytokine and the gene family of tyronase were investigated based on the culture of melanocytes in vitro. The most practical study is to find some drugs which are safe and effective to regulate the melanocytes and melagenesis. Retinoids are used abroad now and with much pharmacological activity. The effect of it on skin color is found in clinic.
We investigated the effects of retinoic acid and other drugs on B16 murine melanoma cells and normal human melanocytes in vitro, in search of the most effective concentration and get insight on the mechanisms involved. B16 murine melanoma cells and normal human melanocytes were cultured in vitro. The cells from the same original bottle were transplanted to the 96 well cell culture cluster. The cell concentration were about 5*105/ml. B16 murine melanoma cells were incubated for 24 hours, while the normal human melanocytes were incubated for 48 hours. Then the culture medium was replaced and retinoic acid or other drugs
were added to it. After treating for 72 hours, tyrosinase activity, melanin content and cell proliferation rate were detected.
The results showed that retinoic acid exhibited an inhibitory effect on tyrosinase activity and melanin production at high concentration (>500 μmol/L), and could promote the cell proliferation. Retinoic acid and hydroquinone could be cooperative at high concentration(>500μmol/L), enhanced the down regulation of tyrosinase activity and melanin content. Retinoic acid could also mitigate the inhibitory effect of hydroquinone on cell proliferation, so as to protect the cells from injure. Retinoic acid showed slight inhibitory effect on the up regulation of tyrosinase activity and melanin content which were induced by TPA,IBMX and CT,it could promote the cell proliferation with IBMX and CT over 1000 μ mol/L.
引文
1, Kim-NS; Behavioral differences between donor site-matched adult and neonatal melanocytes in culture. Arch-Dermatol-Res. 2000 May; 292(5) : 233-9
2, Schallreuter-KU Downregulation of tyrosinase activity in human melanocyte cell cultures by yohimbine J-Invest-Dermatol. 2000 Jul; 115(1) : 130-1
3, Ranson-M; Extracellular matrix modulates the function of human melanocytes but not melanoma cells. J-Cell-Physiol. 1988 Aug, 136(2) : 281-8
4,雷铁池等 甘草酸、熊果苷及氢醌对小鼠黑素瘤细胞黑素生成影响的比较研究 临床皮肤科杂志 2000, 29 (2) : 69-72
5, Abdel-Naser-MB Differential effects on melanocyte growth and melanization of low vs. high calcium keratinocyte-conditioned medium. Br-J-Dermatol. 1999 Jan; 140(1) : 50-5
6, Calcitonin gene-related peptide upregulates melanogenesis and enhances melanocyte dendricity via induction of keratinocyte derived melanotrophic factors. J-Investig-Dermatol-Symp-Proc. 1999 Sep;4(2) : 116-25
7, Jake Gittlen Molecular mechanisms controlling human melanoma progression and metastasis. Pathobiology. 1997; 65(6) : 311-30
8, Goding-CR Regulation of melanocyte differentiation and growth. Cell-Growth-Differ. 1997 Sep; 8(9) : 935-40
9, Krasagakis-K Growth control of melanoma cells and melanocytes by cytokines. Recent-Results-Cancer-Res. 1995; 139: 169-82
10, alpha-MSH and the regulation of melanocyte function. Ann-N-Y-Acad-Sci. 1999 Oct 20; 885: 217-29
11, Molecular mechanisms controlling human melanoma progression and metastasis. Pathobiology. 1997; 65(6) : 311-30
12, Regulation of melanocyte differentiation and growth Cell-Growth-Differ. 1997 Sep, 8(9) : 935-40
13, PigmentaTIon and proliferaTIon of human melanocytes and the effects of melanocyte-sTImulaTIng hormone and ultraviolet B light. Ann-N-Y-Acad-Sci. 1993 May 31; 680: 290-301
14, Evidence for nerve growth factor-mediated paracrine effects in human epidermis. J-Cell-Biol. 1991 Nov; 115(3) : 821-8
15, The biology of the melanocyte Prog-Clin-Biol-Res. 1988; 256: 463-74
16, Basic fibroblast growth factor from human keraTInocytes is a natural mitogen for melanocytes. J-Cell-Biol. 1988 Oct; 107(4) : 1611-9
17, Human epithelial cells induce human melanocyte growth in vitro but only skin keraTInocytes regulate its proper differenTIaTIon in the absence of dermis. J-Cell-Biol, 1988 Nov; 107(5) : 1919-26
18, Cell-density-dependent regulation of expression and glycosylation of dopachrome tautomerase/tyrosinase-related protein-2． J-Invest-Dermatol. 2000 Jul; 115(1) : 106-12
19, Yoshimura H, et al. A novel type of retinoic acid receptor antagonist: synthesis and structure-activity relationships of heterocyclic ring-containing benzoic acid derivatives. J Med Chem, 1995;38(16) : 3163-3173
20, Johnson AT et al. Synthesis and characterization of a highly potent and effective antagonist of retinoic acid receptors. J Med Chem, 199538(24) :4764-4767
21, Rosen J et al. Intracellular receptors and signal transducers and activators of transcription superfamilies: novel targets for small-molecule drug
discovery, J Med Chem, 1995;38(25) :4855-4874
22, Lehmann JM et al. Retinoids selective for retinoid X receptor response pathways. Science, 1992;258(5090) : 1944-1946
23, Variations in melanin formation by cultured melanocytes from different skin types. Arch-Dermatol-Res. 1998 Jun; 290(6) : 342-9
24, Mapping of the human gene for a melanocyte protein Pmel 17 (D12S53E) to chromosome 12q13-q14． Genomics. 1995 Mar 20; 26(2) : 430-1
25, KeraTInocytes and fibroblasts in a human skin equivalent model enhance melanocyte survival and melanin synthesis after ultraviolet irrADiaTIon. J-Invest-Dermatol. 1995 May; 104(5) : 859-67
26, Growth of human melanocyte cultures supported by 12-O-tetrADecanoylphorbol-13-acetate is mediated through protein kinase C acTIvaTIon. Cancer-Res. 1992 Aug 15; 52(16) : 4514-21
27, 雷铁池 与表皮黑素细胞增殖和黑素生成有关的信号转导 国外医学皮肤性病学分册 1999; 25: 32-35
28, Regulatory mechanisms of melanogenesis: beyond the tyrosinase concept. J-Invest-Dermatol. 1993 Feb; 100(2 Suppl): 156S-161S
29, Role of tyrosinase as the determinant of pigmentaTIon in cultured human melanocytes. J-Invest-Dermatol. 1993 Jun; 100(6) : 806-11
30, Russell M et al. J Invest Dermatol 1996,1(2) : 119-122
31, Expression of integrin receptors and their role in ADhesion, spreADing and migraTIon of normal human melanocytes. J-Cell-Sci. 1993 May; 105 (Pt 1) : 179-90
32, Co-culture of human melanocytes and keraTInocytes in a skin equivalent model: effect of ultraviolet rADiaTIon. Arch-Dermatol-Res. 1993, 285(8) : 455-9
33, Growth and differenTIaTIon of normal human melanocytes in a
TPA-free, cholera toxin-free, low-serum medium and influence of keraTInocytes. Arch-Dermatol-Res. 1993; 285(7): 385-92
34、赵辨等 体外传代培养人正常黑素细胞的电镜观察 临床皮肤科杂志 1993；3：124-126
35、Protein kinase C: biochemical characterisTIcs and role in melanocyte biology. J-Dermatol-Sci. 1993 Dec; 6(3): 185-93
36、赵辨等 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；5：226-228
37、Eisinger Met al. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc Natl Acad Sci USA 1982;79:2018
38、Halaban R et al. Human melanocytes cultured from nevi and melanomas. J Invest Dermatol 1986,87:95
39、A study of the effects of phorbol 12-myristate-13-acetate on cell differenTIaTIon of pure human melanocytes in vitro. Arch-Dermatol-Res. 1991; 283(2): 119-24
40、Growth characteristics of human epidermal melanocytes in pure culture with special reference to geneTIc differences. J-Cell-Physiol. 1988 May; 135(2): 262-8
2, Schallreuter-KU Downregulation of tyrosinase activity in human melanocyte cell cultures by yohimbine J-Invest-Dermatol. 2000 Jul; 115(1) : 130-1
3, Ranson-M; Extracellular matrix modulates the function of human melanocytes but not melanoma cells. J-Cell-Physiol. 1988 Aug, 136(2) : 281-8
4,雷铁池等 甘草酸、熊果苷及氢醌对小鼠黑素瘤细胞黑素生成影响的比较研究 临床皮肤科杂志 2000, 29 (2) : 69-72
5, Abdel-Naser-MB Differential effects on melanocyte growth and melanization of low vs. high calcium keratinocyte-conditioned medium. Br-J-Dermatol. 1999 Jan; 140(1) : 50-5
6, Calcitonin gene-related peptide upregulates melanogenesis and enhances melanocyte dendricity via induction of keratinocyte derived melanotrophic factors. J-Investig-Dermatol-Symp-Proc. 1999 Sep;4(2) : 116-25
7, Jake Gittlen Molecular mechanisms controlling human melanoma progression and metastasis. Pathobiology. 1997; 65(6) : 311-30
8, Goding-CR Regulation of melanocyte differentiation and growth. Cell-Growth-Differ. 1997 Sep; 8(9) : 935-40
9, Krasagakis-K Growth control of melanoma cells and melanocytes by cytokines. Recent-Results-Cancer-Res. 1995; 139: 169-82
10, alpha-MSH and the regulation of melanocyte function. Ann-N-Y-Acad-Sci. 1999 Oct 20; 885: 217-29
11, Molecular mechanisms controlling human melanoma progression and metastasis. Pathobiology. 1997; 65(6) : 311-30
12, Regulation of melanocyte differentiation and growth Cell-Growth-Differ. 1997 Sep, 8(9) : 935-40
13, PigmentaTIon and proliferaTIon of human melanocytes and the effects of melanocyte-sTImulaTIng hormone and ultraviolet B light. Ann-N-Y-Acad-Sci. 1993 May 31; 680: 290-301
14, Evidence for nerve growth factor-mediated paracrine effects in human epidermis. J-Cell-Biol. 1991 Nov; 115(3) : 821-8
15, The biology of the melanocyte Prog-Clin-Biol-Res. 1988; 256: 463-74
16, Basic fibroblast growth factor from human keraTInocytes is a natural mitogen for melanocytes. J-Cell-Biol. 1988 Oct; 107(4) : 1611-9
17, Human epithelial cells induce human melanocyte growth in vitro but only skin keraTInocytes regulate its proper differenTIaTIon in the absence of dermis. J-Cell-Biol, 1988 Nov; 107(5) : 1919-26
18, Cell-density-dependent regulation of expression and glycosylation of dopachrome tautomerase/tyrosinase-related protein-2． J-Invest-Dermatol. 2000 Jul; 115(1) : 106-12
19, Yoshimura H, et al. A novel type of retinoic acid receptor antagonist: synthesis and structure-activity relationships of heterocyclic ring-containing benzoic acid derivatives. J Med Chem, 1995;38(16) : 3163-3173
20, Johnson AT et al. Synthesis and characterization of a highly potent and effective antagonist of retinoic acid receptors. J Med Chem, 199538(24) :4764-4767
21, Rosen J et al. Intracellular receptors and signal transducers and activators of transcription superfamilies: novel targets for small-molecule drug
discovery, J Med Chem, 1995;38(25) :4855-4874
22, Lehmann JM et al. Retinoids selective for retinoid X receptor response pathways. Science, 1992;258(5090) : 1944-1946
23, Variations in melanin formation by cultured melanocytes from different skin types. Arch-Dermatol-Res. 1998 Jun; 290(6) : 342-9
24, Mapping of the human gene for a melanocyte protein Pmel 17 (D12S53E) to chromosome 12q13-q14． Genomics. 1995 Mar 20; 26(2) : 430-1
25, KeraTInocytes and fibroblasts in a human skin equivalent model enhance melanocyte survival and melanin synthesis after ultraviolet irrADiaTIon. J-Invest-Dermatol. 1995 May; 104(5) : 859-67
26, Growth of human melanocyte cultures supported by 12-O-tetrADecanoylphorbol-13-acetate is mediated through protein kinase C acTIvaTIon. Cancer-Res. 1992 Aug 15; 52(16) : 4514-21
27, 雷铁池 与表皮黑素细胞增殖和黑素生成有关的信号转导 国外医学皮肤性病学分册 1999; 25: 32-35
28, Regulatory mechanisms of melanogenesis: beyond the tyrosinase concept. J-Invest-Dermatol. 1993 Feb; 100(2 Suppl): 156S-161S
29, Role of tyrosinase as the determinant of pigmentaTIon in cultured human melanocytes. J-Invest-Dermatol. 1993 Jun; 100(6) : 806-11
30, Russell M et al. J Invest Dermatol 1996,1(2) : 119-122
31, Expression of integrin receptors and their role in ADhesion, spreADing and migraTIon of normal human melanocytes. J-Cell-Sci. 1993 May; 105 (Pt 1) : 179-90
32, Co-culture of human melanocytes and keraTInocytes in a skin equivalent model: effect of ultraviolet rADiaTIon. Arch-Dermatol-Res. 1993, 285(8) : 455-9
33, Growth and differenTIaTIon of normal human melanocytes in a
TPA-free, cholera toxin-free, low-serum medium and influence of keraTInocytes. Arch-Dermatol-Res. 1993; 285(7): 385-92
34、赵辨等 体外传代培养人正常黑素细胞的电镜观察 临床皮肤科杂志 1993；3：124-126
35、Protein kinase C: biochemical characterisTIcs and role in melanocyte biology. J-Dermatol-Sci. 1993 Dec; 6(3): 185-93
36、赵辨等 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；5：226-228
37、Eisinger Met al. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc Natl Acad Sci USA 1982;79:2018
38、Halaban R et al. Human melanocytes cultured from nevi and melanomas. J Invest Dermatol 1986,87:95
39、A study of the effects of phorbol 12-myristate-13-acetate on cell differenTIaTIon of pure human melanocytes in vitro. Arch-Dermatol-Res. 1991; 283(2): 119-24
40、Growth characteristics of human epidermal melanocytes in pure culture with special reference to geneTIc differences. J-Cell-Physiol. 1988 May; 135(2): 262-8