驱虫斑鸠菊对B-16黑素瘤细胞酪氨酸酶活性及黑素生成影响

目的 :探讨驱虫斑鸠菊体外对酪氨酸酶活性影响 ,以及对小鼠B - 16黑素瘤细胞株细胞增殖、黑素合成以及细胞内酪氨酸酶的作用。方法 :利用四甲基偶氮唑蓝 (MTT)比色法测定药物对细胞增殖的影响 ;采用酶学方法研究药物对酪氨酸酶活性的影响 ;470nm比色法测定黑素含量。结果驱虫斑鸠菊体外可激活酪氨酸酶活性 ,增强B - 16鼠黑素瘤细胞增殖 ,提高酪氨酸酶和黑色素合成能力 ;对整体动物黑素细胞具有促进合成和分泌作用。结论在白癜风的治疗中 ,驱虫斑鸠菊可增强酪氨酸酶活性 ,进而促进黑素合成     

驱虫斑鸠菊注射液治疗白癜风的作用机制研究

探讨驱虫斑鸠菊(VW)注射液治疗白癜风的作用机制。进行以下实验探讨VW对小鼠免疫功能的影响:淋巴细胞转化试验测定小鼠脾T、B细胞增殖活性;脾细胞介导羊红细胞定量溶血分光光度法测定B细胞生成抗体活性,流式细胞法测定B细胞上CD19表达活性;迟发型超敏反应(DTH)试验测定T细胞活性、眼3H演-TdR掺入法测定T细胞分泌IL-2活性等。用酶学方法研究VW对小鼠体内酪氨酸酶的作用。用半定量逆转录聚合酶链反应技术检测酪氨酸酶基因表达活性。结果表明,VW可以明显抑制小鼠体内T、B细胞的增殖反应(P<0.01);对绵羊红细胞(SRBC)诱导的正常小鼠脾脏抗体形成细胞活性、CD19B细胞亚类表达、小鼠DTH反应和T细胞分泌IL-2活性也具有明显的抑制作用,这些抑制作用与药物浓度有一定的剂量效应关系。VW还可以提高小鼠血清酪氨酸酶活性,增强酪氨酸酶基因的表达。以上结果说明,VW可抑制小鼠免疫功能,可以从转录水平增强酪氨酸酶活性,进而促进黑素合成。     

二苯乙烯苷对黑素细胞黑素合成及酪氨酸酶活性的影响

为了研究何首乌有效成分二苯乙烯苷对黑素合成及黑素细胞酪氨酸酶活性的影响。采用体外培养的正常人表皮黑素细胞作为实验对象解法测定二苯乙烯苷作用72 h后的黑素细胞黑素含量;多巴氧化法测定二苯乙烯苷作用72 h后的黑素细胞酪氨酸酶活性。研究发现,3组不同浓度的药物对黑素合成产生了不同的影响。两组低浓度药物作用下黑素含量与对照组比较有所增加;一组较高浓度的药物抑制了黑素的合成。3组药物对酪氨酸酶活性也有不同影响,与对照组相比两组较低浓度的药物提高了实验对象酪氨酸酶的活性;而高浓度的药物对酪氨酸酶无明显的抑制或提高。     

驱虫斑鸠菊对淋巴细胞亚类的影响

探讨驱虫斑鸠菊注射液对小鼠免疫功能的影响,揭示其免疫作用机理。采用流式细胞技术测定驱虫斑鸠菊注射液对小鼠脾脏淋巴细胞亚类表达的影响。结果表明,驱虫斑鸠菊可以增强CD4、CD8、CD3T细胞分化抗原的表达,抑制CD19 B细胞分化抗原的表达;说明驱虫斑鸠菊可增强细胞免疫功能、抑制体液免疫功能。     

驱虫斑鸠菊对小鼠免疫分子的影响

探讨驱虫斑鸠菊对小鼠免疫功能的影响,揭示其免疫作用机理。利用[^3H]-TdR参入法测定驱虫斑鸠菊对小鼠体内免疫功能的影响,运用酶联免疫吸附实验测定驱虫斑鸠菊对小鼠B淋巴细胞分泌抗体功能的影响;采用流式细胞测定方法测定CD19B细胞亚类表达水平;采用[^3H]-TdR参入法利用CTLL－2细胞株测定T淋巴细胞分泌IL－2活性。结果驱虫斑鸠菊的低、中、高三个剂量对体内T、B淋巴细胞的增殖活性、血清总抗体和抗原特异性抗体含量、CD19B细胞亚类表达均有明显的抑制作用,对T淋巴细胞分泌IL－2活性也具有明显抑制作用。说明驱虫斑鸠菊对机体体液免疫和细胞免疫功能都具有明显抑制作用。     

他卡西醇对人黑素细胞生物学性状的影响

目的:观察他卡西醇对体外培养的正常人黑素细胞增殖、黑素合成及酪氨酸酶(TYR)mRNA表达、酪氨酸酶相关蛋白-1(TRP-1)mRNA表达的影响,探讨他卡西醇治疗白癜风的机制.方法:体外黑素细胞培养,给予不同浓度他卡西醇(10-6、10-7、10-8、10-9、10-10mol/L)处理,用MTT法测定细胞增殖,碱裂解法测定黑素合成,多巴氧化法检测酪氨酸酶活性,半定量RT-PCR法检测TYR、TRP-1 mRNA的表达.结果:10-8 mol/L他卡西醇能刺激黑素细胞增殖,10-8、10-9 mol/L能促进黑素合成,10-9～10-6 mol/L的他卡西醇能显著增强正常人黑素细胞酪氨酸酶活性并上调TYR、TRP-1mRNA的表达.结论:他卡西醇可促进黑素细胞增殖,并提示该药物可能通过上调酪氨酸酶家族中关键基因TYR、TRP-1mRNA的表达来提高酪氨酸酶活性,最终增加黑素合成.     

环状RNA Circ_0001073在恶性黑素瘤中的表达及其对A375细胞恶性表型的影响

目的:通过检测环状RNA Circ_0001073在恶性黑素瘤细胞与正常表皮黑素细胞中的表达差异,及其对恶性黑素瘤A375细胞恶性表型的影响,阐明Circ_001073在恶性黑素瘤细胞中的表达及功能。方法:采用聚合酶链式反应(Real-time quantitative polymerase chain reaction, RT-QPCR)方法检测正常表皮黑素细胞和恶性黑素瘤细胞中Circ_0001073的表达水平;应用小干扰RNAs沉默恶性黑素瘤A375细胞中Circ_0001073表达,5-乙炔基-2'-脱氧尿苷(5-Ethynyl-2'-deoxyuridine,EdU)实验检测沉默Circ_0001073对A375细胞增殖的影响,平板克隆实验检测沉默Circ_0001073对A375细胞克隆形成的影响,细胞划痕实验评估沉默Circ_0001073对A375细胞迁移能力的影响,Transwell实验评估沉默Circ_0001073对A375细胞侵袭能力的影响。结果:qRT-PCR结果显示Circ_0001073在恶性黑素瘤细胞中的表达低于正常表皮黑素HEMa-LP细胞(P0.05)。应用Circ_0001073si RNA转染A375细胞后,Circ_0001073的表达水平较转染Circ_0001073 NC的细胞显著降低(P0.05)。EdU实验结果显示:沉默A375细胞中Circ_0001073的表达后,A375细胞增殖能力显著增强(P0.05);平板克隆实验结果显示:沉默A375细胞中Circ_0001073的表达后,A375细胞克隆形成能力显著增强(P0.05);细胞划痕实验结果显示:沉默A375细胞中Circ_0001073的表达后,细胞迁移能力显著增强(P0.05);Transwell实验结果显示:沉默A375细胞中Circ_0001073的表达后,细胞侵袭能力显著增强(P0.05)。结论:Circ_0001073在恶性黑素瘤细胞中低表达,并可抑制A375细胞的增殖、克隆形成、迁移及侵袭。     

低氘水对皮肤成纤维细胞及黑色素瘤细胞的影响

研究不同浓度的低氘水对正常皮肤成纤维细胞CCD-1095sk增殖和乳酸代谢的影响,及对B16黑色素瘤细胞酪氨酸酶活性和黑色素生成量的影响。同时,观察低氘水对紫外损伤细胞修复的作用。用MTT法检测25、50和105 ppm三种不同浓度的低氘水对CCD-1095sk细胞增殖的影响,并测定培养液中的乳酸含量;取对数生长期小鼠B16黑素瘤细胞,分别加入不同浓度的低氘水作用72 h后,测定黑素含量和酪氨酸酶活性;体外氧化多巴反应方法测定酪氨酸酶活性;用不同浓度的低氘水作用经紫外辐射的皮肤成纤维细胞,测定细胞增殖率。在细胞培养初期,50 ppm和105 ppm低氘水可促进CCD-1095sk细胞生长,同时抑制乳酸代谢。体外生化实验表明,三种不同浓度的低氘水均能不同程度地抑制酪氨酸酶活性。细胞实验表明,作用72 h后,50 ppm和105 ppm低氘水能显著抑制酪氨酸酶活性,减少B16黑色素瘤细胞黑色素的生成量,差异具有统计学意义,P<0.05。紫外损伤实验表明,50 ppm低氘水能减轻紫外辐射对细胞增殖的抑制作用,差异具有统计学意义,P<0.05。低氘水在化妆品应用方面具有潜在的价值。     

人源性Rab38荧光融合蛋白表达载体的构建及功能鉴定

目的构建人源性Rab38与EGFP融合蛋白表达载体pEGFP-Rab38,观察Rab38在人黑素细胞瘤细胞系A375中的细胞定位情况,为进一步研究Rab38在黑素代谢中的作用提供实验依据。方法收集A375细胞,提取总RNA并逆转录成cDNA。特异性引物PCR扩增Rab38片段并插入pEGFP-C3载体,随后转化至感受态大肠杆菌DH5α中,挑取单克隆菌落进行鉴定。将鉴定正确的重组表达载体转染A375细胞,检测细胞中Rab38和Tyrp1(酪氨酸酶相关蛋白1)的表达并观察Rab38的亚细胞定位。结果 pEGFP-Rab38重组质粒经菌液PCR、双酶切和测序鉴定正确;转染A375细胞后,激光共聚焦显微镜观察到Rab38定位于胞浆中,且大量聚集于细胞核周围;实时定量PCR和Western blot可检测出细胞中EGFP-Rab38融合蛋白的表达,且细胞中Tyrp1的表达明显升高。结论融合蛋白表达载体pEGFP-Rab38成功构建,观察了Rab38在细胞中的定位并初步研究了其对黑素合成关键蛋白Tyrp1的影响,为进一步研究Rab38在黑素代谢中的作用奠定了良好的基础。     

The effects of histamine H2 receptor antagonists on melanogenesis and cellular proliferation in melanoma cells in culture

B16-C3 murine melanoma, A375P human melanotic melanoma, and C32 human amelanotic melanoma cells were incubated in the presence of (0-4 mM) H2-antagonists, ranitidine and cimetidine. Cell proliferation, tyrosinase activity and melanin content were monitored. H2-antagonists stimulated tyrosinase activity and melanin accumulation in B16-C3 cells in a dose- and time-dependent manner. Stimulation of enzyme activity and pigment production was accompanied by inhibition of cellular proliferation in B16-C3 cells. The inhibitory concentration of cimetidine was approximately 2-fold higher than that of ranitidine. H2-antagonists failed to stimulate melanogenesis in A375P or C32 cells, but inhibited cellular proliferation in both cell lines. These results are the first demonstration of H2-antagonist induced phenotypic changes in malignant melanoma cells in vitro, and represent a novel mechanism for the previously described in vivo antitumor effects of these agents.     

Phytochemical potential of Daphne gnidium in inhibiting growth of melanoma cells and enhancing melanogenesis of B16-F0 melanoma

In this study, we have investigated inhibitory capacity of ethyl acetate, total oligomer flavonoid (TOF), aqueous extracts and beta amyrin acetate, a triterpene isolated from ethyl acetate extract obtained from leaves of Daphne gnidium, on mouse melanoma (B16-F0 and B16-F10 cells) proliferation. Influence of these products on percentage cell distribution in cycle phases and melanogenesis was also studied. Cell viability was determined using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and flow cytometry was used to analyse effects of tested compounds on progression through the cell cycle. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Ethyl acetate, TOF and aqueous extracts exhibited significant anti-proliferative activity after incubation with the two types of tumour skin cells B16-F0 and B16-F10. Furthermore, cell cycle analysis revealed that cells treated with ethyl acetate and TOF extracts were arrested predominantly in G2-M phase. Ethyl acetate extract has also the ability to enhance melanogenesis and tyrosinase activity of B16-F0 melanoma cells. Copyright © 2012 John Wiley & Sons, Ltd.     

Chloroform extract from Moricandia arvensis inhibits growth of B16-F0 melanoma cells and promotes differentiation in vitro

Objectives: Poor therapeutic results have been reported for treatment of malignant melanoma; therefore in this study we have investigated inhibitory capacity of ethyl acetate, chloroform (Chl) and methanol extracts from Moricandia arvensis on mouse melanoma (B16‐F0) and human keratinocyte (HaCaT) cell proliferation. Influence of Chl extract on percentage distribution in cell cycle phases and melanogenesis was also studied. Material and methods: Cell viability was determined at various periods using the MTT assay, and flow cytometry was used to analyse effects of Chl extract on progression through the cell cycle and apoptosis. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Results: Chl extract exhibited significant anti‐proliferative activity after incubation with the two types of tumour skin cells. Morphological changes in B16‐F0 cells, accompanied by increase of tyrosinase activity, and of melanin synthesis were observed, which are markers of differentiation of malignant melanoma cells. Furthermore, cell cycle analysis revealed that B16‐F0 cells treated with Chl extract were arrested predominantly in G₁ phase. Conclusion: Chl extract had the ability to reverse malignant melanoma cells from proliferative to differentiated state, thus providing a new perspective in developing novel strategies for prevention and treatment of malignant melanoma, possibly through consumption of the extract in an appropriate cancer prevention diet. Moreover, there is scope for the extract being introduced into cosmetic products as a natural tanning agent.     

Anisaldehyde, a melanogenesis potentiator

Anisaldehyde (4-methoxybenzaldehyde), previously reported as a tyrosinase inhibitor, did not inhibit melanogenesis in cultured B16-F10 melanoma cells but rather enhanced it. This adverse effect of anisaldehyde was accompanied by melanocytotoxicity in a dose-dependent manner up to 2 mM. The melanin content per cell at 1 mM was increased 5-fold compared to control and morphological observations showed the deposition of melanin pigments. Anisaldehyde was also examined against cultured human A375 melanoma cells.     

Effects of radix polygoni multiflori components on tyrosinase activity and melanogenesis

Radix Polygoni multiflori is a herb used effectively to prevent graying and treat skin depigmentation diseases in traditional Chinese medicine but its active ingredients have not been discovered yet. In this investigation, we tested six compounds isolated from Radix Polygoni multiflori, to discover the active component on melanogenesis. Three experiments were performed in the present investigation: mushroom tyrosinase activity, melanin content B16 cell proliferation assay. Among all the six components tested, THSG showed the most potent effects on tyrosinase activation and melanogenesis; it was shown to be a potent tyrosinase activator and a melanogenesis stimulator in this study. On the other hand, we found that gallic acid significantly inhibited tyrosinase and, in addition, anthraquinones were cytotoxic to melanoma cells. They were both harmful to melanogenesis. Therefore, we propose that THSG acts as the active ingredient of Radix Polygoni multiflori on melanogenesis.