七白膏方剂中药提取物对离体人黑素细胞的作用

目的:评价七白膏方剂中的药物对离体培养人黑素细胞的作用.方法:建立体外人表皮黑素细胞模型.醇提法提取各种中药活性成分.以熊果苷为实验对照,检测各种中药提取液对于离体培养人黑素细胞增殖、酪氨酸酶活性及黑素合成的干预作用.结果:低浓度白芷对黑素细胞增殖呈抑制作用(P＜0.05),但高浓度时略呈激活作用.其它中药对黑素细胞增殖和酪氨酸酶活性有抑制作用.不同中药提取液对于黑素合成抑制作用不同.结论:各种中药提取液可影响黑素细胞增殖、酪氨酸酶活性和黑素合成,从而干预体外人黑素细胞的代谢.     

复方中药对小鼠B－16黑素瘤细胞株黑素合成和酪氨酸酶的激活作用

目的 研究复方中药对小鼠 B－ 16黑素瘤细胞株细胞增殖、黑素合成的影响,及其对细胞内酪氨酸酶的激活作用。方法 四甲基偶氮唑蓝 (MTT)比色法测定中药对细胞增殖的影响;酶学方法研究中药对酪氨酸酶活性的作用; 470 nm比色标准曲线法测定黑素含量。结果 研究发现复方 3号（补肝肾为主）促进黑素细胞增殖和色素合成的能力高于复方 1号（光敏感性药物）或复方 2号（活血化瘀）,而酶激活能力复方 1号较为稳定。结论 在白癜风治疗中补肝肾类中药不可忽视。同时也说明影响黑素合成的因素是多方面的,可能不仅仅与酪氨酸酶活性有关。     

血清药理学方法研究复方中药对B16黑素瘤细胞黑素合成的影响

目的运用血清药理学方法研究复方中药白斑一号对小鼠B16黑素瘤细胞黑素合成的影响.方法体外培养小鼠B16黑素瘤细胞,运用血清药理学、细胞学的方法,测定药物对B16黑素瘤细胞增殖情况,对酪氨酸酶活性调节作用及黑素含量的影响.结果复方中药白斑一号能促进黑素合成和激活酪氨酸酶,从而阐明了该方在白癜风治疗中的机制.     

胡椒碱等6种中药单体促进黑素瘤Cloudman S91细胞株黑素合成的研究

目的研究胡椒碱、原儿茶醛、白果内脂等6种中药单体对小鼠黑素瘤细胞黑素合成的影响,初步探讨不同的中药单体影响黑素合成的作用机制。方法培养的黑素瘤CloudmanS91细胞分别以系列浓度的6种中药单体作用72h,测定细胞黑素含量、酪氨酸酶活性并与体外蘑菇酪氨酸酶活性直接测定方法(多巴速率氧化法)测定的结果进行比较。结果6种单体均能促进CloudmanS91细胞的黑素量生成。胡椒碱、原儿茶醛、白果内脂在最佳促进浓度均较100μmol/L的8-甲氧补骨脂素作用明显(P<0.05),其中0.5mmol/L胡椒碱对黑素含量促进作用最为明显。在实验浓度(0.01～1.00mmol/L)胡椒碱可促进酪氨酸酶活性,体外可直接增加蘑菇酪氨酸酶的活性;原儿茶醛对酪氨酸酶活性无明显影响,体外可直接促进蘑菇酪氨酸酶的活性;白果内脂对酪氨酸酶活性起促进作用,体外对蘑菇酪氨酸酶的活性无影响。结论中药单体胡椒碱、原儿茶醛、白果内脂可明显促进黑素瘤细胞黑素的合成,原儿茶醛的作用可能不依赖于酪氨酸酶的活化作用。     

桃红四物汤对酪氨酸酶活性与黑素生成的影响

目的：观察桃红四物汤对酪氨酸酶活性与黑素生成的影响。方法：采用直接添加法和血清药理学方法，观察待测物对酪氨酸酶活性及黑素生成的影响；蘑菇酪氨酸酶多巴速率氧化法体外测定酪氨酸酶活性，氢氧化钠溶解法检测黑素生成量。结果：桃红四物汤加减水煎剂和药物血清均有明显激活酪氨酸酶活性的作用，并呈剂量依赖性。水煎剂呈现明显的促黑素生成活性。结论：桃红四物汤加减在体内外均具有激活酪氨酸酶作用，且体外有促黑素生成活性。     

中药含药血清对体外培养黑素细胞增殖及酪氨酸酶活性的影响

目的探讨中药补肾化瘀胶囊含药血清对黑素细胞增殖及酪氨酸酶活性的影响。方法体外培养人黑素细胞株,制备大鼠中药含药血清,分别采用四甲基偶氮唑蓝比色法(MTT)和蘑菇酪氨酸酶多巴速率氧化法测定黑素细胞增殖及酪氨酸酶活性。结果中药含药血清均能促进黑素细胞增殖,与空白对照组差异有显著性(P<0.05);三组含药血清间两两比较差异无显著性(P>0.05)。补肾化瘀胶囊组较六味地黄丸组、逍遥丸组、空白对照组有显著抑制酪氨酸酶活性的作用,差异有显著性(P<0.01);六味地黄丸组、逍遥丸组、空白对照组三组两两比较,差异均无显著性(P>0.05)。结论补肾化瘀胶囊含药血清对黑素细胞增殖具有促进作用,对酪氨酸酶活性具有明显抑制作用,在促进黑素细胞增殖上各组中药含药血清作用相当,在抑制酪氨酸酶活性上补肾化瘀胶囊组优于六味地黄丸组和逍遥丸组。     

中药滋补肝肾方对人黑素细胞株黑素合成的影响

目的通过中药滋补肝肾方对人黑素细胞株细胞增殖、酪氨酸酶活性和黑素含量影响的研究,探讨本方治疗白癜风的作用机制.方法采用4-甲基偶氮唑蓝比色法(MTT法)测定本方对细胞增殖的影响,用酶学方法测定对酪氨酸酶活性的影响,比色法测定中药对黑素含量的影响.结果滋补肝肾方对体外培养的人黑素细胞具有促进细胞增殖、提高黑素含量和酪氨酸酶活性作用.结论本方能促进黑素细胞黑素合成,这可能是其治疗白癜风的作用机理之一.     

六种中药复方乙醇提取物对酪氨酸酶激活作用及动物致色素作用的研究

目的探讨中医理法和实验药效的关系及其可能的作用机制,为临床治疗白癜风优化组方提供实验依据。方法离体实验为中药复方乙醇提取物对酪氨酸酶的影响;动物实验选用棕色豚鼠为模型,观察中药致色素作用。结果Ⅰ方、Ⅱ方、Ⅲ方、Ⅵ方对酪氨酸酶有激活作用(P<0.05);6种中药复方均使豚鼠表皮基底层中含黑素颗粒细胞增多(P<0.05);Ⅱ方、Ⅲ方、Ⅵ方中药使豚鼠表皮内Dopa阳性细胞增多(P<0.05)。结论对酪氨酸酶具有激活作用的中药组成复方并不一定对酪氨酸酶有激活作用;中药复方在离体和活体上的作用并不完全平行,提示中药对黑素生成的机理,不仅限于对酪氨酸酶的激活作用。     

白癜风丸系列对酪氨酸酶活性影响的实验研究

采用蘑菇酪氯酸酶多巴速率氧化法测定白癜风丸系列对酪氨酸酶活性的影响。白癜风丸系列能上调酪氨酸酶的活性，其中1、3号几与对照组相比有显著性差异（P〈0．01）。提示这些中药可能通过调节酪氨酸酶活性促进黑素合成治疗白癜风。     

中药桂枝抑制黑素生成的作用机理研究

目的研究中药桂枝对小鼠黑素细胞系M el-a黑素生成、酪氨酸酶活性及基因表达的影响,阐明桂枝抑制黑素生成的机理。方法用药物处理M el-a细胞后进行黑素含量、L-DOPA染色、放免法测定酪氨酸酶活性,western b lot和实时RT-PCR分别测酪氨酸酶及酪氨酸酶相关蛋白的蛋白和mRNA表达水平。选取熊果苷作为阳性对照。结果10μg/m l浓度的桂枝提取物在细胞培养水平对黑素生成有明显地抑制作用,强于20μg/m l浓度的熊果苷。桂枝可以抑制酪氨酸酶mRNA表达;减少这种限速酶的蛋白产量,在细胞量相同的情况下,桂枝明显抑制酪氨酸酶的氧化活性。结论中药桂枝有很强的抑制黑素产生的作用,其作用是通过抑制酪氨酸酶的基因表达、蛋白合成和氧化活性这三方面来实现。     

赤芍等5种上调酷氨酸酶活性中药乙醇提取液对棕黄色豚鼠背部皮肤的增色作用

为了研究中药对黑素细胞生物学特性的影响,筛选有增色作用的中药,为应用中药治疗白癜风等色素减退性皮肤病提供实验诊据。选定体外对酷氨酸酶活性有显著激活作用的中药5味,用中药乙醇提取液外涂棕黄色豚鼠背部皮肤,然后取材,进行HE、Schmorl、Imokawa等方面染色,观察外用中药后黑素细胞数量和形态的改变。结果,赤芍、川芎、菟丝子、补骨脂、刺蒺藜等中药组多巴阳性黑素细胞数、含黑素颗粒细胞数及黑素含量指数均较空白对照组明显增多（P＜0.01或P＜0.05),其中菟丝子、赤芍组与阳性对照组0.1%8-MOP酊外用无显著性差异（P＞0.05)。提示赤芍、川芎、菟丝子、补骨脂、刺蒺藜等中药具有增色作用,临床上可试用于白癜风等色素减退性皮肤病。     

47种中药对酪氨酸酶活性的影响及酶动力学的研究

目的:研究47种治疗白癜风常用中药对酪氨酸酶的影响。方法:通过体外实验观察中药乙醇提取物对酪氨酸酶的激活作用及对酶动力学的影响。结果:19种中药的乙醇提取物能显著激活酪氨酸酶的活性,其中鸡血藤、夏枯草、女贞子、薄荷、潼蒺藜、申姜、旱莲草、黄芩、泽兰、甘草和山(毛)慈姑对酪氨酸酶的激活作用均明显高于补骨脂素;中药对酪氨酸酶的激活作用表现为竞争性、非竞争性和混合性激活作用。结论:部分中药治疗白癜风有效与其激活酪氨酸酶有关,具有混合性激活作用的中药对酶活性影响较大。     

芪白合剂对黑素细胞作用的研究

目的：研究芪白合剂对黑素细胞的影响，探讨其治疗白癜风的药理作用。方法：将兔含药血清加入传代培养的正常人黑素细胞，用MTT法、多巴氧化法及NaOH法测定含药血清对黑素细胞的增殖、酪氨酸酶的活性及黑素合成的影响。结果：未灭活组含药血清可以促进黑素细胞增殖，上调酪氨酸酶活性以及增加黑素合成，效果较灭活组显著。结论：芪白合剂直接作用于黑素细胞，促进黑素细胞增殖，上调酪氨酸酶的活性，增加黑素合成，可用于白癜风的治疗。     

Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes

Background: There is a need to develop an in vitro skin models which can be used as alternative system for research and testing pharmacological products in place of laboratory animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo, reliable in vitro skin pigmentation models are required. Aim: In this study, we used primary cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and vitiligo patients. Methods: The skin grafts were taken from control and patients of vitiligo. In vitro co-culture was prepared after culturing primary melanocytes and keratinocytes. Co- cultures were treated with melanogenic stimulators and inhibitors and after that tyrosinase assay, MTT assay and melanin content assay were performed. Results: Melanocytes and keratinocytes were successfully cultured from control and vitiligo patients and after that co-culture models were prepared. After treatment of co-culture model with melanogenic stimulator we found that tyrosinase activity, cell proliferation and melanin content increased whereas after treatment with melanogenic inhibitor, tyrosinase activity, cell proliferation and melanin content decreased. We also found some differences in the control co-culture model and vitiligo co-culture model. Conclusion: We successfully constructed in vitro co-culture pigmentation model for control and vitiligo patients using primary cultured melanocytes and keratinocytes. The use of primary melanocytes and keratinocytes is more appropriate over the use of transformed cells. The only limitation of these models is that these can be used for screening small numbers of compounds.     

FK506 increases pigmentation and migration of human melanocytes

BACKGROUND: Topical tacrolimus (FK506) is a potential therapeutic option for vitiligo management. Despite its clinical efficacy, the underlying mechanism of how topical tacrolimus induces repigmentation in vitiligo has scarcely been investigated. OBJECTIVES: To investigate the direct effects of FK506 on pigmentation and migration of human melanocytes. METHODS: Cell proliferation was measured using a Coulter counter. The effects on pigmentation were investigated by measuring melanin contents, tyrosinase activity and tyrosinase expression. To determine the effects of FK506 on cell migration, we performed scratch assays and Boyden chamber assays. RESULTS: FK506 treatment increased melanin contents, although there was an inhibitory effect on growth of melanocytes. The increase of pigmentation was due to the result of the stimulatory action of FK506 on tyrosinase activity and its expression, which eventually led to melanin biosynthesis. Furthermore, cell migration was enhanced by FK506 treatment. CONCLUSIONS: These findings provide in vitro evidence demonstrating direct effects of FK506 on pigmentation and melanocyte migration and may provide a possible mechanism for the effect of tacrolimus in vitiligo.     

Narrow‐band UVB irradiation stimulates the migration and functional development of vitiligo‐IgG antibodies‐treated pigment cells

Background The pathogenesis of vitiligo remains unclear. Most authorities favoured the autoimmune cause for the strong associations of vitiligo with multiple autoimmune diseases and the presence of autoantibodies in vitiligo patients. Narrow‐band UVB (NBUVB) irradiation has been considered to be an effective treatment for vitiligo with simple treatment procedure and decreased accumulated ultraviolet exposure doses. Objectives The aim this study was to investigate the effects of NBUVB irradiation on normal IgG antibodies (N‐IgG) or vitiligo IgG antibodies (V‐IgG)‐treated NCCmelan5 cells in terms of proliferation, migration and melanin formation. Methods Cultured NCCmelan5 cells were treated with (i) NBUVB irradiation alone, (ii) N‐IgG or V‐IgG alone, and (iii) combination of N‐IgG or V‐IgG with NBUVB irradiation. The proliferation of NCCmelan5 cells were evaluated using BrdU incorporation assay. Western blotting was used to determine the expressions of phosphorylated p125^FAK (pp125^FAK) and tyrosinase in NCCmelan5 cells. The locomotion of NCCmelan5 cells was assessed using time‐lapse assay and in vitro wound scratch assay. Results Neither N‐IgG nor V‐IgG significantly affected the proliferation of NCCmelan5 cells. The migration, melanin formation and tyrosinase expression in NCCmelan5 cells were decreased by V‐IgG. NBUVB irradiation increased the proliferation of V‐IgG treated NCCmelan5 cells. In addition, NBUVB irradiation enhanced the mobility of V‐IgG‐treated NCCmelan5 cells via upregulation of pp125^FAK. The melanogenesis and tyrosinase expression in V‐IgG‐treated NCCmelan5 cells were promoted using NBUVB irradiation. Conclusions Our study demonstrated that the deleterious effects of V‐IgG in the pathogenesis of vitiligo might be overcome by NBUVB irradiation.     

槲皮素对鼠黑素细胞株B10BR细胞的抗氧化保护效应及对其生物学活性的影响

目的 探讨槲皮素保护鼠永生化黑素细胞（B10BR）对抗氧化应激的有效性及其对B10BR细胞生物学活性影响。方法 MTT法测定B10BR细胞存活率,流式细胞仪检测细胞凋亡率,倒置显微镜下观察细胞形态改变,并检测槲皮素对酪氨酸酶活性及黑素合成的影响。结果 经33.33 μmol/L槲皮素预处理24 h后细胞活性增高至（94.22 ± 3.36）%,此外黑素细胞的酪氨酸酶活性及黑素含量可分别增高至（107.15 ± 10.96）%和（111.85 ± 9.49）%,与过氧化氢处理组相比,差异均有统计学意义（P < 0.01）。流式细胞仪检测结果表明,槲皮素可抑制过氧化氢诱导的黑素细胞凋亡。结论 槲皮素抑制过氧化氢诱导黑素细胞凋亡的保护效应为其治疗白癜风提供一定的依据。