【作者】 张秉新；

【导师】 刘全忠；

【作者基本信息】 天津医科大学 ， 皮肤性病学， 2002， 硕士

【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。更多还原

【Abstract】 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 drugswere 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.更多还原