加味桃红四物汤治疗白癜风的临床及实验室研究

目的 观察加味桃红四物汤治疗白癜风的效果并探讨其作用机制。方法 白癜风患者243例，用加味桃红四物汤口服液，每次10mL，3次／d，口服。体外培养的正常人黑素细胞以含药血清作用后测定细胞酪氨酸酶活性、黑素含量和细胞增殖。结果 临床观察显示243例白癜风患者服用加味桃红四物汤后，治愈26例(10．7％)，显效66例(27．2％)，有效76例(31．3％)，无效75例(30．8％)；实验结果显示加味桃红四物汤的含药血清能上调酪氨酸酶活性，提高黑素含量，促进黑素细胞增殖。结论加味桃红四物汤治疗白癜风有明显疗效，其作用可能与刺激黑素细胞黑素合成、上调酪氨酸酶活性、促进细胞增殖有关。     

龙胆草在人表皮黑素细胞与角质形成细胞共培养体系中对酪氨酸酶活性及黑素生成影响

目的：探讨龙胆草对人表皮黑素细胞与角质形成细胞共培养体系酪氨酸酶活性及黑素生成的影响。方法：培养人表皮黑素细胞与角质形成细胞共培养体系,利用四甲基偶氮唑蓝（MTT）还原法测定细胞活力,采用酶学方法测定酪氨酸酶活性,475nm比色法测定黑素含量。结果：0．1mg／mL、0．2mg／mL和0．5mg／mL龙胆草对于共培养细胞酪氨酸酶活性有较强的剂量相关性激活作用（P〈0．01）,0．2mg／mL及0．5mg／mL龙胆草与对于共培养细胞黑素生成有较强的剂量相关性激活作用（P〈0．01）。结论：龙胆草对于共培养体系酪氨酸酶活性及黑素生成有较强的剂量相关性激活作用。     

复方中药白癜冲剂对B16黑素瘤细胞生物学特性影响的实验研究

目的：观察复方中药白癜冲剂对体外培养的B16黑素瘤细胞生物学特性的影响。方法：体外培养B16黑素瘤细胞,测定不同浓度白癜冲剂对B16黑素瘤细胞增殖、对酪氨酸酶活性和黑素含量的影响。结果：低浓度剂量组白癜冲剂对B16黑素瘤细胞较对照组有明显促增殖作用,黑素合成增多,酪氨酸酶活力增加,差异均有统计学意义（P〈0．05）。结论：复方中药白癜冲剂能促进B16黑素瘤细胞的黑素合成,提高酪氨酸酶活力。     

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。     

欧前胡素抑制人表皮黑素细胞黑素生成

目的：评价欧前胡素对培养人表皮黑素细胞黑素生成的作用。方法：体外分离、纯化培养人表皮黑素细胞。随机分为空白组、熊果苷组和欧前胡素组（12．5μM,25μM和50μM）,作用48h,观察黑素细胞形态．检测黑素细胞活力、酪氨酸酶活性和黑素含量,免疫荧光显微镜观察欧前胡素对黑素细胞酪氨酸酶的影响。结果：不同浓度（0、12．5μM、25μM和50μM）欧前胡素作用于黑素细胞48h后,细胞活力分别为100％、107．8％、87．5％和59．7％。低浓度欧前胡素不影响黑素细胞形态,高浓度（50μM）对黑素细胞活力、酪氨酸酶活性、黑素含量有抑制作用,呈剂量依赖性。欧前胡素对酪氨酸酶蛋白在细胞内定位未见明显影响,但酪氨酸酶荧光强度降低。结论：欧前胡素抑制体外培养人表皮黑素细胞酪氨酸酶活性而降低黑素生成。     

芪归祛白胶囊治疗小鼠白癜风的实验研究

目的:确定芪归祛白胶囊治疗白癜风的药效学机制。方法:组织病理学观察黑素的变化;应用蘑菇酪氨酸酶多巴速率氧化法体外测定酪氨酸酶活性;通过碳粒廓清功能的变化明确其对单核巨噬细胞吞噬功能的影响。结果:白癜风动物模型经芪归祛白胶囊治疗后皮肤棘层及表皮基底细胞黑素分布及有黑素毛囊数均明显增加;酪氨酸酶活性明显增高;单核巨噬细胞对血清中异物的吞噬功能明显降低。结论:芪归祛白胶囊对白癜风小鼠模型有治疗作用。     

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。     

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.     

内皮素-1对培养的黑素细胞生物学特性的影响

目的:探讨内皮素-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.方法:采用不含佛波醇酯和霍乱毒素的培养液培养黑素细胞,倒置显微镜观察细胞形态,分别用CCK-8法、多巴氧化法和NaOH法测定细胞增殖、酪氨酸酶活性和黑素含量.结果:内皮素-1可促进黑素细胞的树突形成,可上调酪氨酸酶活性,增加黑素合成,均在10 nM 时最显著.1～100 nM的内皮素-1可明显刺激细胞增殖,随时间延长效应更明显.结论:一定浓度的内皮素-1具有促进培养的人黑素细胞树突形成、增殖、上调酪氨酸酶活性和增加黑素生成的作用.     

山奈素对melan-a黑素细胞株黑素生成的影响

目的:观察山奈素对melan-a黑素细胞株黑素生成的影响。方法:山奈素干预melan-a后,观察对黑素细胞增殖率的影响并测定各组酪氨酸酶活性大小及黑素量。结果:山奈素表现出轻度的促melan-a增殖作用(P<0.01),当山奈素浓度在0.001mmol/L至0.1mmol/L之间时,细胞增殖率和浓度呈指数关系,而浓度高于0.1mmol/L后进入平台期。山奈素在1mmol/L浓度时,促黑素合成的作用与同浓度8-MOP类似(P>0.05)。Fontana-Masson染色后与空白对照比较,经山奈素干预的细胞内,黑素颗粒染成黑色,融合成片,明显多于空白对照。结论:山奈素能够激活melan-a黑素细胞酪氨酸酶的活性,并具有促进黑素细胞增殖、使黑素合成量增加的作用。     

Pigment-lightening effect of N,N'-dilinoleylcystamine on human melanoma cells

BACKGROUND: Cystamine and linoleic acid have been reported to reduce melanin synthesis in vitro and in vivo. N,N'-dilinoleylcystamine (DLC) is a compound of cystamine and linoleic acid connected by an ester bond. OBJECTIVES: To investigate the effects of DLC on melanin synthesis using cultured human melanoma cells. METHODS: Levels of total melanin, eumelanin and phaeomelanin, tyrosinase protein and tyrosinase activity in situ were measured in HM3KO melanoma cells. Changes in degree of pigmentation were quantified by image analysis and compared with absorbance values. Tyrosinase from HM3KO cells was used to measure the direct effect of DLC on DOPA and DOPAchrome production. RESULTS: At concentrations of 1.4-14 micromol L-1, DLC reduced the pigmentation of HM3KO melanoma cells but did not affect cell growth. The visual decrease in pigmentation produced by DLC was more dramatic than the decrease in total melanin content as measured by absorbance at 500 nm. DLC treatment decreased eumelanin synthesis and increased phaeomelanin synthesis in HM3KO melanoma cells. An in situ tyrosinase assay showed that DLC inhibited tyrosinase activity, as well as the level of tyrosinase protein. CONCLUSIONS: These results suggest that DLC has pigment-lightening effects on HM3KO melanoma cells, produced by reducing the level of eumelanin while increasing the level of phaeomelanin.     

苦参碱及肉桂酸对体外培养小鼠黑素细胞黑素生成的影响

目的:观察苦参碱和肉桂酸对melan-a小鼠黑素细胞黑素生成的影响。方法:用药物干预melan-a小鼠,观察黑素细胞的形态并测定各组黑素量及酪氨酸酶的活性。结果:肉桂酸干预melan-a小鼠黑素细胞2d后就表现出明显的促进树突生成的作用。肉桂酸和苦参碱在高、中、低浓度(1.00、0.10、0.01mmol/L)有抑制melan-a小鼠黑素细胞酪氨酸酶活性的作用(除外0.01mmol/L苦参碱),其抑制作用均弱于对照药氢醌(P<0.01)。苦参碱(0.001～1.000mmol/L)和肉桂酸(0.001～1.000mmol/L)均能使melan-a小鼠黑素细胞黑素量减少,但其抑制黑素合成的作用明显低于同浓度氢醌。Fontana-Masson染色表明,经苦参碱干预后的melan-a小鼠黑素细胞内黑素颗粒少于空白对照组。结论:苦参碱和肉桂酸均有抑制melan-a小鼠黑素细胞酪氨酸酶活性的作用及使黑素合成量减少的作用,且苦参碱作用强于肉桂酸,而肉桂酸有促进黑素细胞树突形成的作用。     

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。     

The superoxide anion may mediate short- but not long-term effects of ultraviolet radiation on melanogenesis

Abstract The present study was carried out to examine the role of reactive oxygen species in mediating the melanogenic effects of UVR. B16 mouse melanoma cells responded to a single dose of UVR by showing increases in their melanin content. Although there was a small increase in melanin at 48-72 hours, which was associated with a rise in tyrosinase activity at 48 h, the greatest change occurred at 3 h and this was not associated with an increase in tyrosinase activity. This short-term response, unlike the more delayed melanogenic response, was reduced by superoxide dismut-ase (SOD). Xanthine oxidase (XO), which generates the superoxide anion (O₂). also increased the melanin content of B16 melanoma cells with effects at 3 h and 48 h. As with UVR, the delayed response was accompanied by an increase in tyrosinase activity but no such association was evident at 3 h. In addition, the short-term effect, like that seen with UVR, was reduced with SOD and to a lesser extent with catalase. In contrast to the effects found with XO, glucose oxidase, which generates hydrogen peroxide, had no effect on the melanin content or tyrosinase activity of the B16 cells. These results confirm previous observations that UVR is able to act directly on cells to bring about delayed increases in melanogenesis. They further demonstrate that UVR also stimulates melanogenesis through a more rapid action that is not associated with an activation of tyrosinase. This effect could be mediated by the O₂ which, rather than activating tyrosinase, could act by serving as a substrate for the enzyme.     

The influence of hormones on melanogenesis

The contribution of melanin to the colour of the skin is subject to hormonal regulation. Hormones influence the amount and the distribution of melanin in the epidermis. One mechanism by which hormones can influence the amount of melanin involves changes in the activity of the enzyme tyrosinase in the melanoblasts which synthesize the pigment. A second mechanism may involve changes in the activity of keratinocytes which engulf the melanin discharged from melanoblasts. Hormones can also influence the dispersion of melanin. Interstitial cell-stimulating hormone, oestrogens, melanocyte-stimulating hormone and adrenocorticotrophin appear to increase epidermal melanin by enhancing the activity of tyrosinase. Hormones probably exert other effects on epidermal melanin which are less clearly understood. An attempt is made to point out the immediate limitations in our present understanding of the contribution of melanin to the colour of the skin and directions of future investigations are suggested.     

芦荟素对Melan-a鼠黑素细胞株黑素生成及其相关基因表达的影响

目的:研究芦荟素对Melan-a鼠黑素细胞株的黑素生成及其相关基因表达的影响。方法:分别测定不同浓度芦荟素作用后的Melan-a鼠黑素细胞株的细胞增殖率、酪氨酸酶(TRY)含量及黑素量变化,逆转录-聚合酶链反应(RT-PCR)测定酪氨酸酶相关蛋白(TRP)-1和TRP-2的表达,并与相应浓度的氢醌进行比较。结果:0.01～10.00mmol/L的芦荟素对黑素细胞的增殖率无影响,芦荟素可显著抑制Melan-a鼠黑素细胞株的TRY活性和黑素量生成,抑制作用随芦荟素的浓度升高而增加,0.10mmol/L芦荟素处理的Melan-a鼠黑素细胞株的TRY抑制率及黑素量与0.10μmol/L的氢醌相当(P>0.05);芦荟素还能显著下调TRP-1和TRP-2 mRNA的表达水平(P<0.01),但是TRP-1和TRP-2的变化与TRY及黑素量的变化不平行。结论:芦荟素显著抑制Melan-a鼠黑素细胞株的TRY活性、黑素量和TRP-1/TRP-2 mRNA的表达水平,直接测定黑素细胞TRY浓度和黑素量比检测TRP-1/TRP-2mRNA更能确切地反映芦荟素对黑素细胞的影响。     

山柰素与熊果苷对体外培养人正常黑素细胞的作用

目的比较山柰素与熊果苷对体外培养人正常黑素细胞增殖、黑素合成及其酪氨酸酶活性的影响,为探索色素沉着性皮肤病的发病机制以及筛选新的治疗药物提供理论依据。方法以不同浓度(1～100μmol/L)山柰素与熊果苷干预体外培养的人正常黑素细胞,比较两者对黑素细胞的黑素含量、细胞增殖及其酪氨酸酶活性的影响。结果对于黑素细胞酪氨酸酶活性和黑素含量的抑制作用,山柰素在各个浓度均优于熊果苷,差异有统计学意义(P<0.05),山柰素对细胞增殖抑制率无明显影响,而熊果苷在各浓度对细胞增殖抑制作用显著,且随其浓度增加,抑制率逐渐增强。结论山柰素对黑素细胞酪氨酸酶活性及黑素合成的抑制作用强于熊果苷,并对黑素细胞的增殖及形态无明显的负面影响,是一种有良好应用前景的酪氨酸酶抑制剂。