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。     

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.     

疏肝活血中药复方对B16小鼠黑素瘤细胞黑素合成的影响

目的探讨疏肝活血中药复方治疗黄褐斑的作用机制.方法以系列浓度的疏肝活血中药复方水煎剂作用于体外培养的B16小鼠黑素瘤细胞,测定细胞酪氨酸活性、黑素含量和细胞增殖率,琼脂糖凝胶电泳检测细胞凋亡.结果疏肝活血中药复方水煎剂能剂量依赖性抑制体外培养的B16小鼠瘤细胞酪氨酸活性和黑素合成,抑制细胞增殖,但无细胞凋亡发生.结论疏肝活血中药复方可直接作用于黑素细胞,抑制细胞黑素合成,但无细胞毒作用,可用于治疗黄褐斑.     

N-乙酰氨基葡萄糖对酪氨酸酶活性及小鼠B16黑素瘤细胞黑素合成的影响

目的 探讨N-乙酰氨基葡萄糖对体外酪氨酸酶活性、小鼠B16黑素瘤细胞酪氨酸酶和黑素合成的影响。方法 取对数生长期小鼠B16黑素瘤细胞,分别加入不同浓度N-乙酰氨基葡萄糖（1.5、3、6.25、25、50和100 mmol/L）共孵育,MTT法测定培养的小鼠B16黑素瘤细胞的增殖活性,NaOH法测定黑素生成量,体外氧化多巴反应方法测定酪氨酸酶活性;免疫细胞化学测定小鼠B16黑素瘤细胞中酪氨酸酶的表达。结果 与阴性对照（0.8183 ± 0.0326）相比,1.5、3、6.25、25和50 mmol/L N-乙酰氨基葡萄糖显著抑制蘑菇酪氨酸酶活性（分别为0.7380 ± 0.0254、0.7293 ± 0.0382、0.7247 ± 0.0389、0.7233 ± 0.0186、0.7043 ± 0.0166）（P < 0.05或P < 0.01）;25、50和100 mmol/L N-乙酰氨基葡萄糖显著抑制小鼠B16黑素瘤细胞增殖（分别为0.5410 ± 0.0496、0.4480 ± 0.0246、0.1273 ± 0.0137）和黑素生成（0.0070 ± 0.0008、0.0049 ± 0.0012、0.0015 ± 0.0014）,50和100 mmol/L N-乙酰氨基葡萄糖显著抑制B16黑素瘤细胞酪氨酸酶活性（分别为0.1003 ± 0.0404、0.0130 ± 0.0053）及酪氨酸酶蛋白的表达（13.2700 ± 0.9741、8.5667 ± 2.0345,P < 0.05或P < 0.01）。结论 N-乙酰氨基葡萄糖抑制培养的小鼠B16黑素瘤细胞的酪氨酸酶活性和黑素合成。     

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。     

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。     

维A酸对A375细胞黑素合成及酪氨酸酶蛋白表达的影响

目的探讨维A酸对人A375黑素瘤株细胞黑素含量、酪氨酸酶活性及蛋白表达的影响。方法将培养的A375细胞分为维A酸组、8-甲氧补骨脂素(8-MOP)组、氢醌组、阴性对照组(A375细胞只加入相应溶媒,不加入药物)分别培养,于24 h、48 h和72 h测定黑素含量及酪氨酸酶活性;培养24 h Western检测酪氨酸酶蛋白的变化。结果 A375细胞加入维A酸24 h、48 h、72 h后黑素含量及酪氨酸酶活性均较阴性对照组下降(P0.05)。阴性对照组A375细胞酪氨酸酶蛋白含量测定值为(0.89±0.085),维A酸组A375细胞酪氨酸酶蛋白含量为(0.56±0.044)(t=3.811,P0.05)。结论维A酸能抑制人A375黑素瘤株细胞黑素含量、酪氨酸酶活性及蛋白表达。     

CLEC2B基因过表达的Jurkat细胞培养上清液对黑素瘤细胞B16的影响

目的将CLEC2B基因过表达的Jurkat细胞培养上清液作用于黑素瘤细胞,观察其上清液对黑素瘤细胞增殖、酪氨酸酶活性、黑素合成的影响,探讨CLEC2B基因在白癜风发病中的作用。方法培养人淋巴瘤细胞Jurkat细胞和小鼠黑素瘤细胞B16,采用脂质体介导的方法瞬时转染CLEC2B重组质粒入Jurkat细胞,半定量RT-PCR法鉴定CLEC2B基因过表达;将其细胞培养上清液作用于黑素瘤细胞48 h后,MTT法检测黑素瘤细胞的增殖情况,多巴氧化法检测酪氨酸酶活性,氢氧化钠裂解法检测黑素含量。结果 CLEC2B基因过表达的Jurkat细胞培养上清液作用后的黑素瘤细胞增殖比空载体组、正常对照组降低,差异有统计学意义(均P0.05),CLEC2B过表达组黑素含量比空载体组、正常对照组减少,差异有统计学意义(均P0.05),CLEC2B过表达组、空载体组、正常对照组之间的酪氨酸酶活性比较差异无统计学意义(均P0.05),空载体组与正常对照组比较差异均无统计学意义(均P0.05)。结论 CLEC2B基因过表达的Jurkat细胞培养上清液对黑素细胞的增殖和黑素合成有一定的抑制作用,对酪氨酸酶活性影响不明显。提示CLEC2B可能通过调节淋巴细胞功能间接影响黑素细胞增殖和黑素合成,从而参与白癜风发病。     

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.     

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.     

18味中药乙醇提取物对小鼠B16黑素瘤细胞增殖及黑素生成的影响

目的研究中药乙醇提取物对小鼠B16黑素瘤细胞增殖及黑素合成的影响。方法以MTT法测定培养的B16黑素瘤细胞的增殖活性:NaOH法测定黑素生成量;体外氧化多巴反应方法测定酪氨酸酶活性。结果与空白对照组相比,18种中药乙醇提取物中,有9种提取物对1种以上指标有显著性作用(P<0.05或P<0.01)。沙苑子、甘草、山慈姑、薄荷、苦参、夏枯草、黄芩、黑芝麻、墨旱莲可明显促进B16黑素瘤细胞增殖,甘草、山慈姑、薄荷、苦参、夏枯草、何首乌、黑芝麻、墨旱莲明显增加黑素含量,山慈姑、薄荷、苦参、夏枯草、黑芝麻、墨旱莲明显增加酪氨酸酶活性,山慈姑、薄荷、苦参、夏枯草、黑芝麻和墨旱莲的提取物对3种指标均有显著性作用(P<0.05或P<0.01)。结论山慈姑等6种中药的乙醇提取物具有促进黑素细胞增殖和合成黑素的作用。     

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.     

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.