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.     

复方木尼孜其颗粒对小鼠B16黑素瘤细胞酪氨酸酶活性及黑素合成的影响

目的 研究复方木尼孜其颗粒对小鼠B16黑素瘤细胞酪氨酸酶活性及黑素合成的影响.方法 培养小鼠B16黑素瘤细胞,采用MTT法测定不同浓度复方木尼孜其颗粒对细胞增殖的影响,采用L-DOPA氧化法测定其对细胞酪氨酸酶活性的影响,采用NaOH裂解法检测细胞黑素含量.结果 复方木尼孜其颗粒高剂量组能明显抑制小鼠B16黑素瘤细胞黑素的合成,但是对酪氨酸酶活性无影响.结论 复方木尼孜其颗粒能抑制小鼠B16黑素瘤细胞黑素的合成,在治疗色素性皮肤病中有一定前景.     

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。     

复方甘草酸苷注射液对B16鼠黑素瘤细胞黑素生成的影响

目的：研究复方甘草酸苷注射液对B16鼠黑素瘤细胞黑素的影响及其机制。方法：通过CCK8、流式细胞术、NaOH裂解法、多巴氧化法和Western-blotting技术检测复方甘草酸苷对B16鼠黑素瘤细胞生长,黑素合成与转运及酪氨酸酶活性和蛋白含量的影响。结果：(1) 复方甘草酸苷注射液浓度低于30μl/ml,对B16鼠黑素瘤细胞生长无明显影响;浓度大于50μl/ml,细胞生长速率减慢;浓度达到500μl/ml,细胞生长明显受抑制。(2) 浓度在10μl/ml到200μl/ml之间,黑素的合成和酪氨酸酶的活性均增加。黑素的合成在复方甘草酸苷注射液浓度为50μl/ml时增加最明显;且酪氨酸酶活性呈浓度依赖性增加,在浓度为100μl/ml和150μl/ml时达到最高。结论：复方甘草酸苷注射液有促进黑素瘤细胞黑素的合成与转运及增加酪氨酸酶活性的作用。     

积雪草甙对酪氨酸酶活性的抑制作用及对Cloudman S91黑素瘤细胞黑素合成的影响

目的探讨积雪草甙外用脱色素的作用机制.方法体外观察积雪草甙对蘑菇酪氨酸酶活性的作用并进行酶促反应动力学分析.进一步以系列浓度的积雪草甙作用于体外培养的Cloudman S91黑素瘤细胞,测定细胞酪氨酸酶活性、黑素含量和细胞增殖率.结果积雪草甙能剂量依赖性抑制蘑菇酪氨酸酶活性,酶促动力学分析提示积雪草甙为蘑菇酪氨酸酶混合型抑制剂.进一步研究发现,积雪草甙能抑制体外培养的Cloudman S91黑素瘤细胞酪氨酸酶活性和黑素合成,但对细胞增殖无明显影响.与氢醌相比,积雪草甙抑制酪氨酸酶活性低于相同浓度的氢醌.结论积雪草甙可直接作用于黑素细胞,抑制黑素合成,是一种无细胞毒性的皮肤脱色剂.     

内皮素拮抗剂对鼠黑素瘤细胞黑素生成影响的研究

目的 研究天然提取的内皮素拮抗剂对体外培养的B16鼠黑素瘤细胞的生物学作用。方法比较观察了内皮素拮抗剂和甘草黄酮对体外培养的B16鼠黑素瘤细胞酪氨酸酶活性、黑素含量和细胞增殖率的影响,以及内皮素拮抗剂对内皮素（ET-1）引起的B16细胞酪氨酸酶活性变化的作用。结果 在实验浓度下,甘草黄酮具有浓度依赖性黑素合成抑制作用,内皮素拮抗剂对培养的B16黑素瘤细胞黑素生成没有直接的抑制作用,但能特异性抑制内皮素对黑素瘤细胞分化和酪氨酸酶的激活作用,200 μg/mL该拮抗剂即可显著拮抗0.5 μg/mL ET-1对黑素瘤细胞的刺激增殖作用,与甘草黄酮相比,细胞毒性较小。结论 内皮素拮抗剂是一种安全的皮肤美白物质,在UVB照射后内皮素增加引起的皮肤色素沉着中,具有广泛的应用前景。     

中药消白灵汤对黑素细胞影响的实验研究

目的：观察中药消白灵汤对体外培养的鼠黑素细胞的影响，探讨中药消白灵汤治疗白癜风的疗效机制。方法：用MTT法测定中药消白灵汤及其主要成分不同浓度对体外培养的鼠B16F10黑素瘤细胞系黑素细胞生长增殖的情况；用NaOH裂解法测定中药消白灵汤及其主要成分不同浓度对黑素细胞黑素合成的影响。结果：消白灵汤组、补肾阳药组和养血活血药组及其不同浓度在第3天至第5天均能明显促进黑素细胞增殖（P〈0.05），且以消白灵汤组浓度为1mg／mL者刺激黑素细胞的增殖更明显（P〈0．01）；补肾阳药组浓度为2mg／mL和3mg／mL者和养血活血药组浓度为3mg／mL者对黑素细胞合成黑素的能力均有所增强（P〈0.05）。结论：中药消白灵汤能促进黑素细胞增殖和黑素合成，并提示补肾阳药和养血活血药在治疗白癜风的组方用药中的重要作用。     

全反式维甲酸和阿达帕林对中波紫外线诱导的melan-a黑素细胞黑素合成的影响

目的 探讨外用维甲酸脱色素作用的机制.方法 利用体外培养的melan-a黑素细胞,观察全反式维甲酸和阿达帕林对中波紫外线(UVB)诱导的melan-a黑素细胞黑素合成的影响.结果 二者可剂量依赖性抑制UVB诱导的细胞酪氨酸酶活性和黑素合成,使其回复到基础水平,但对基础黑素合成无明显影响,10-5M的全反式维甲酸可抑制黑素细胞增殖,阿达帕林对细胞增殖无明显影响.全反式维甲酸和阿达帕林对细胞黑素合成的影响无明显差异.结论提示维甲酸可直接作用于黑素细胞抑制其黑素合成,这可能是维甲酸脱色素作用的机制之一.     

淫羊藿苷等6种中药单体抑制Cloudman S91黑素瘤细胞株黑素合成的研究

观察淫羊藿苷、白藜芦醇苷、红景天苷等6种中药单体对体外培养的CloudmanS91黑素瘤细胞黑素合成和酪氨酸酶活性的影响。方法:选择系列浓度梯度的中药单体作用于体外培养的CloudmanS91黑素瘤细胞,测定细胞酪氨酸酶活性、黑素含量和细胞增殖率。结果:淫羊藿苷、白黎芦醇苷和红景天苷3种药物明显抑制酪氨酸酶活性和黑素的生成(P<0.05),且呈浓度依赖性,作用3d内呈时间依赖性。其中淫羊藿苷的作用与氢醌比较,差异无显著性(P>0.05),而白黎芦醇苷和红景天苷的作用明显弱于氢醌(P<0.01)。淫羊藿苷、白黎芦醇苷和红景天苷均促进细胞的增殖,其中淫羊藿苷对细胞增殖促进作用明显。结论:淫羊藿苷、白黎芦醇苷和红景天苷能抑制CloudmanS91黑素瘤细胞黑素合成和酪氨酸酶活性。     

白消一号方对黑素瘤细胞与角质形成细胞混合培养模型中黑素合成的影响

目的 体外构建黑素瘤细胞与角质形成细胞直接接触的混合培养模型,观察白消一号方对此模型中黑素瘤细胞的影响,探讨其治疗白癜风的药理作用.方法 分别培养角质形成细胞、黑素瘤细胞;体外构建黑素瘤细胞与角质形成细胞直接接触的混合培养模型,大鼠中药灌胃后不同时间取血清加入此模型中,用MTT法、多巴氧化法、及NaOH法.检测含药血清对黑素瘤细胞增殖、酪氧酸酶活性以及黑素合成的影响.结果 含药血清可促进黑素瘤细胞增殖,上调酪氨酸酶活性.增加黑素合成.结论 成功构建了黑素瘤细胞与角质形成细胞直接接触的混合培养模型,白消一号方作用于此模型对黑素瘤细胞增殖、酪氨酸酶活性及黑素的合成均呈促进作用.     

Inhibitory effect of arbutin on melanogenesis--biochemical study using cultured B16 melanoma cells

Inhibitory effect of arbutin (hydroquinone-beta-D-glucopyranoside) on the melanogenesis was studied biochemically using cultured B16 melanoma cells. The maximum arbutin concentration lacking an inhibitory effect on cell growth was 5 X 10(-5) M. At this concentration, melanin content per cell was decreased significantly to about 39%, compared with that of arbutin untreated cells. Also, tyrosinase activity of arbutin treated cells was decreased significantly. When arbutin was added to B16 melanoma cell suspension, arbutin was not hydrolyzed to liberate hydroquinone. Further, tyrosinase activity in crude preparations from B16 melanoma cells was inhibited by arbutin. From these results, it is suggested that arbutin can inhibit the melanogenesis by affecting not only the synthesis but also the activity of tyrosinase rather than by killing melanocytes B16 melanoma cells. Also, it is suggested that hydroquinone is not responsible for the inhibitory effect of arbutin on the melanogenesis.     

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.     

墨旱莲乙醇提取物动物致色素作用和对小鼠B16黑素瘤细胞黑素生成的影响

目的选择离体实验中对黑色素合成的关键酶—酪氨酸酶(tyrosinase,TYR)有激活作用的墨旱莲(旱莲草),研究其动物致色素作用和对小鼠B16黑素瘤细胞黑素生成及相关基因犤TYR、TYR相关蛋白(TRP-1/2)mRNA犦表达的影响。方法以棕色豚鼠为动物模型,用Schmorl法染色,计数含黑素的细胞;用多巴(DOPA)-氧化酶染色,计算每100个基底细胞中DOPA阳性细胞数。培养的小鼠B16黑素瘤细胞以四甲基偶氮唑盐比色(MTT)法、氢氧化钠(NaOH)法和体外氧化DOPA反应方法分别测定细胞的增殖活性、黑素生成量及TYR活性;逆转录-聚合酶链反应(RT-PCR)测定TYR及其TRP-1/2基因的表达。结果墨旱莲乙醇提取物可使豚鼠表皮基底层中含黑素颗粒细胞增多,使豚鼠表皮内DOPA阳性细胞增多(P<0.05);具有促进小鼠B16黑素瘤细胞黑素合成及TYR活性作用(P<0.05),对细胞TYR基因有上调作用,而对TRP-1/2mRNA的表达无明显作用。结论墨旱莲乙醇提取物具有促进黑素合成及上调酪氨酸酶基因表达的作用,对白癜风色素恢复有较好应用和开发的前景。     

Antimelanogenesis effect of Tunisian herb Thymelaea hirsuta extract on B16 murine melanoma cells

Skin pigmentation is the result of melanogenesis that occurs in melanocytes and/or melanoma cells. Although melanogenesis is necessary for the prevention of DNA damage and cancer caused by UV irradiation, excessive accumulation of melanin can also cause melanoma. Thus, we focused on the antimelanogenesis effect of an extract from Thymelaea hirsuta, a Tunisian herb. Murine melanoma B16 cells were treated with T. hirsuta extract, and then cell viability and synthesized melanin content were measured. We found that the T. hirsuta extract decreased the synthesized melanin content in B16 cells without cytotoxicity. Tyrosinase is a key enzyme of melanogenesis and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation is known to be related to melanogenesis inhibition. To clarify its mechanism, we also determined ERK1/2 phosphorylation and tyrosinase expression level. ERK1/2 was immediately phosphorylated in cells just after treatment with the extract. The tyrosinase expression was inhibited after 24 h of stimulation with the extract. The T. hirsuta extract was fractionated, and we found that one fraction considerably decreased the melanin synthesis in B16 cells and that this fraction contains daphnanes as the main component. This indicates that our findings might be attributable to daphnanes.     

Effect of Adenosine on Melanogenesis in B16 Cells and Zebrafish

Background

Adenosine is a nucleoside, in which an adenine molecule is attached to a ribofuranose sugar moiety. It can be released into the microenvironment by metabolically active cells, and then fulfills a multitude of functions in regulation of cell proliferation, by activating four subtypes of G protein-coupled adenosine receptors.

Objective

In this study, we investigated the effect of adenosine on melanogenesis, using B16 melanoma cells.

Methods

The toxic effects of adenosine on B16 melanoma cells were assessed. To understand the mechanism of the effect of adenosine on melanogenesis in B16 cells, melanin content and tyrosinase activity were measured. Tyrosinase, tyrosinase-related protein-1, and dopachrome tautomerase were monitored by Western blotting. Finally, adenosine was applied to zebrafish embryos, and its in vivo effect on pigmentation investigated.

Results

At a low concentration, adenosine increased melanin content and tyrosinase activity, while a high dose of adenosine resulted in inhibition of tyrosinase activity. Western blotting showed that adenosine increased tyrosinase protein levels slightly, while high-dose adenosine decreased the expression of tyrosinase. In zebrafish tests, adenosine slightly inhibited body pigmentation.

Conclusion

In this study, we investigated the effect of adenosine on melanogenesis, using the well-established B16 melanoma cell and zebrafish models. The results suggest that adenosine may inhibit pigmentation, through negative regulation of tyrosinase.     

止血环酸抑制黑色素合成的实验研究

目的 观察止血环酸抑制黑素瘤细胞的黑素合成作用并探讨其机理。方法 比色法和免疫组化法分别用于B16黑素瘤细胞黑素含量测定和酪氨酸酶测定，氨基酸自动分析仪和高效液相色谱用于酪氨酸酶代谢的测定。结果 止血环酸虽不能抑制B16黑素瘤细胞生长和减少黑素瘤细胞酪氨酸酶含量，但能干扰酪氨酸酶对酪氨酸的催化作用，减少黑素合成。结论 止血环酸是通过干扰酪氨酸酶的酪氨酸催化作用抑制黑素合成。