CN105190315B - 筛选具有美白功效的物质的方法 - Google Patents
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Abstract
本发明涉及一种筛选具有美白功效的物质的方法,更具体地,涉及一种通过利用作为长寿基因的叉头框蛋白O3a来确认试验物质是否激活叉头框蛋白O3a或促进叉头框蛋白O3a的表达,从而判定试验物质是否具有美白功效的筛选美白功效物质的方法。
Description
技术领域
本发明涉及一种筛选具有美白功效的物质的方法,更具体地,涉及一种通过利用叉头框蛋白O3a(Forkhead box O3A)来确认试验物质是否激活叉头框蛋白O3a或促进叉头框蛋白O3a的表达,从而判定试验物质是否具有美白功效的方法,所述叉头框蛋白O3a为长寿基因叉头框O3(Forkhead box O3)基因所编码的蛋白质。
背景技术
黑色素是决定人皮肤颜色的色素,皮肤的颜色由该黑色素的量和分布而决定。形成黑色素的细胞是在皮肤表皮下的称为黑色素形成细胞(Melanocyte)的细胞中形成,并通过皮肤的新陈代谢移动到角质表面,并脱落。不管皮肤颜色如何,黑色素形成细胞的数量几乎相同。只是由于产生黑色素的量和种类及分布不同,因此皮肤颜色方面有差异。皮肤中的酪氨酸(Tyrosine)通过称为酪氨酸酶(Tyrosinase)的人体酶转化成多巴(DOPA),并通过一系列的氧化过程,最终形成作为黑褐色聚合物的黑色素。
黑色素不能在活体内分解,而是通过角质形成细胞在从表皮脱落时与黑色素一起从皮肤上脱落而被去除。当这种黑色素过度产生时,将诱发诸如黄褐斑、雀斑或痣等的色素过度沉着症,从而对美容方面带来不好的结果,因此,目前对预防因运动等户外活动中的紫外线所引起的黑色素沉着的要求呈增加的趋势。因此,目前的情况为,对于开发一种阻碍黑色素过度形成的美白剂方面的需求正在增大。
因此,寻找这种阻碍黑色素生成而具有美白功效的物质成为重要的问题。需要开发出一种并非通过感官评价或视觉性评价,而是通过客观的基准来确认及判断试验物质的美白功效的方法。
发明内容
要解决的技术问题
对此,本发明人着眼于利用叉头框蛋白O3a能够确认是否具有美白效果这一点,从而完成了本发明,所述叉头框蛋白O3a为长寿基因叉头框O3基因所编码的蛋白质。
因此,本发明的目的在于,提供一种利用叉头框蛋白O3a来筛选具有美白功效的物质的方法。
技术方案
为了实现上述目的,本发明提供一种筛选具有美白功效的试验物质的方法,所述筛选方法包含确认是否激活叉头框蛋白O3a或促进叉头框蛋白O3a的表达。
根据本发明的叉头框蛋白O3a是由已知的长寿基因叉头框O3所编码的蛋白质,并且作为位于胰岛素信号传导通道(IIS,Insulin/IGF-like signaling)的转录因子,是一种对诸如锰超氧化物歧化酶(Mn-SOD)、过氧化氢酶(Catalase)等的酶的表达起作用的蛋白质。如果叉头框蛋白O3a得到激活,则通过体内防御机制的激活来实现抗老化功效。可以通过叉头框蛋白O3a向核的移动来判断所述叉头框蛋白O3a的激活。即,存在于细胞质内的叉头框O3向核移动,从而开始酶的表达。
本发明人确认了根据叉头框蛋白O3a激活的核移动程度和黑色素生成减少程度成比例。因此,通过对皮肤细胞处理测定为具有美白功效的候选物质来确认是否激活叉头框蛋白O3a或促进叉头框蛋白O3a表达,从而能够简单客观地确认所述物质是否为减少黑色素生成而显示出美白功效的物质。
因此,本发明提供一种筛选具有美白功效的物质的方法,其特征为,作为筛选具有美白功效的物质的方法,通过确认试验物质是否激活编码叉头框蛋白O3a(Forkhead boxO3A)蛋白质的基因或促进所述基因的表达来实现。
在本发明的一实施例中,确认所述试验物质是否激活叉头框蛋白O3a的方法可以包含以下步骤:a)使用欲试验的物质对皮肤细胞进行处理;以及观察在所述a)步骤中,经过试验物质处理后的皮肤细胞中的叉头框蛋白O3a的移动。
所述筛选方法可以进一步包含以下步骤:c)如果叉头框蛋白O3a在所述皮肤细胞中向核移动,则判定该物质为具有美白功效的物质。这是因为可以将叉头框蛋白O3a向核的移动视为叉头框蛋白O3a的编码基因得到激活。而且,通过所述筛选方法判定为具有美白功效的物质能够通过叉头框蛋白O3a来开始抗氧化酶的表达,因此,可以判定为显示出抗老化及抗氧化的同时显示出美白功效的物质。
所述步骤a)中的试验物质可以根据试验物质的种类的不同而不同,但是优选以1nM至100nM的浓度进行处理。如果以小于1nM的浓度进行处理,则不能达到试验物质的功效浓度;如果以大于100nM的浓度进行处理,则因过度的功效而细胞稳态会发生异常,反而减少核移动及美白功效。
所述皮肤细胞可以为角质形成细胞、成纤维细胞或黑色素形成细胞。
另外,在本发明的一实施例中,可以通过利用蛋白质印迹法(Western blot)、酶联免疫吸附测定(ELISA)或反转录酶-聚合酶链锁反应(RT-PCR)来确认所述试验物质是否促进叉头框蛋白O3a的表达。
通过所述确认结果,可以判定如果试验物质促进叉头框蛋白O3a的表达,则该物质为具有美白功效的物质。并且,根据所述筛选方法而被判定的具有美白功效的物质从长寿基因叉头框O3的特性方面来说,可以根据叉头框蛋白O3a来开始抗氧化酶的表达,因此,可以判定为不仅具有本发明中发现的美白功效,还可以判定为同时显示出抗老化及抗氧化效果的物质。
有益效果
根据本发明的筛选具有美白功效的物质的方法,由于实际上黑色素形成减少和叉头框蛋白O3a的激活程度成比例,因此可以通过客观性的标准来确认试验物质是否具有美白功效,从而可以不通过临床试验或感官评价等方法,也能够迅速、简单且正确地判断具有美白功效的物质。并且,通过所述筛选方法被判定为具有美白功效的物质,由于可以通过叉头框蛋白O3a来开始抗氧化酶的表达,因此,可以筛选出同时显示出抗老化及抗氧化效果和美白功效的物质。
附图说明
图1a为比较对MNT-1细胞(NT)及MNT-1细胞分别转染野生型叉头框蛋白O3a质粒时(WT),以及转染核位置序列中具有突变的核定位序列(NLS)突变叉头框蛋白O3a质粒时(NLS)的细胞小球(pellet)的溶解液的颜色的照片。
图1b为对MNT-1细胞(NT)及MNT-1细胞分别转染野生型叉头框蛋白O3a质粒时(WT),以及转染核位置序列中具有突变的NLS突变叉头框蛋白O3a质粒时(NLS)的溶解液的蛋白质印迹结果图像。
图2为示出对MNT-1细胞处理维生素C、N-乙酰半胱氨酸及水溶性维生素E(trolox)时的黑色素量和酪氨酸酶活性的图表。
图3为观察对MNT-1细胞处理维生素C、N-乙酰半胱氨酸及水溶性维生素E时的叉头框蛋白O3a的核移动的荧光显微镜照片。
最佳实施方式
以下,将通过实施例及试验例来更加具体地说明本发明的内容。这些实施例仅是为了理解本发明的内容而提出的,本发明的权利范围并不限定于这些实施例及试验例,可以进行所属技术领域中已知的变形、取代及插入等,这些也包含在本发明的范围内。
[试验例1]叉头框蛋白O3a的激活和黑色素生成减少实验
将1X106个MNT-1细胞(人黑色素瘤细胞系,龙沙集团,瑞士)添加到MEM培养基(10%细胞培养基(DMEM))中,并利用脂质体(Lipofectamine)2000(英杰公司)对MNT-1细胞分别转染野生型(WT)叉头框蛋白O3a质粒(Plasmid 1787:HA-叉头框蛋白O3a WT,Addgene公司)和NSL突变叉头框蛋白O3a质粒(nuclear localization sequence mutant plasmid;Plasmid 8361:FLAG-叉头框蛋白O3a TM,Addegene公司),并且在37℃下培养48小时。用500μl的LIPA缓冲液(25mM的三羟甲基氨基甲烷盐酸盐(Tris-HCl),pH7.4,0.1%十二烷基硫酸钠(SDS),0.1%聚乙二醇辛基苯基醚(Triton X-100),1%脱氧胆酸钠(sodiumdeoxycholate),150mM的氯化钠(NaCl),1mM的乙二胺四乙酸(EDTA),1mM的正钒酸钠(Na3VO4),1mM的苯甲基磺酰氟(PMSF),10mg/mL的抑肽酶(aprotinin),5mg/mL亮抑酶肽(leupeptin))溶解(lysis)所述MNT-1细胞后,对细胞溶解物进行离心分离(Centrifuge5415R,艾本德产品,德国),并对获得的小球进行分离,并观察颜色,并且拍摄用200μl的1N氢氧化钠溶解所述小球里的内容物而得到的溶解液的颜色,并在图1a中示出。作为对照组,使用了没有转染质粒的MNT-1细胞。
加载20μg的所述溶解液,并通过蛋白质印迹法来确认叉头框蛋白O3a的蛋白质表达量,将磷酸甘油醛脱氢酶(Gapdh)作为细胞质成分的标记蛋白质使用,以及将核纤层蛋白(Lamin B)作为核成分的标记蛋白质使用,并将结果在图1b中示出。
在图1a的结果中,可以确认转染野生型叉头框蛋白O3a质粒时,由于叉头框蛋白o3a的过表达,因此黑色素的生成显著减少,并且可以确认转染核位置序列中具有突变的NSL叉头框蛋白O3a质粒时,由于叉头框蛋白O3a不会向核移动,因此不会出现黑色素生成减少。另外,从图1b的结果中可以确认,转染野生型叉头框蛋白O3a质粒时,叉头框蛋白O3a得到激活,从而向核移动。因此,可以确认,如果不发生叉头框蛋白O3a的激活,则不会对黑色素生成产生影响,叉头框蛋白O3a向核的移动对黑色素减少起作用。即,可以知道叉头框蛋白O3a的激活为在皮肤细胞中能够有效阻碍黑色素生成的重要因子。
[试验例2]根据美白物质的处理的黑色素生成减少实验
对1X106个的MNT-1细胞(人黑色素瘤细胞系,龙沙集团,瑞士)分别使用0、1nM、10nM、100nM、1μM、10μM、100μM的浓度的已知的美白物质维生素C、N-乙酰半胱氨酸(N-acetyl cysteine;NAC)及水溶性维生素E(Trolox),进行处理后,在37℃下培养48小时。用500μl的LIPA缓冲液溶解(lysis)所述培养的细胞后,对所述细胞溶解质中的500μl进行离心分离(Centrifuge 5415R,艾本德产品,德国),并将获得的小球进行分离,并观察颜色。用200μl的1N氢氧化钠溶解所述小球里的内容物。在对黑色素有特异性的490nm下对所述溶解液的吸光度进行了测定,并利用BCA蛋白浓度测定试剂盒(protein assay kit(Pierce))来测定总蛋白质量。将如此获得的测定值用(吸光度)/(总蛋白质量)进行校准,并用黑色素/μg蛋白质示出黑色素的量。在各美白物质实验组中,计算出将非处理组(0nM)的黑色素量看作1时的实验组的黑色素量的相对值并在图2中示出。
[试验例3]根据处理美白物质的抑制酪氨酸酶活性的评价
在100μl的所述试验例2中的细胞溶解物中添加100μl的10mM多巴(DOPA:dihydroxyphenylalanine),并在37℃下反应1小时后,在490nm下测定吸光度,从而求出由酪氨酸酶的作用而形成的多巴色素(dopachrome)的量。将非处理组(0nM)的吸光度(OD)490值看作1时,计算各实验组的吸光度的相对值,从而求出酪氨酸酶活性抑制能力,并将其在图2中示出。
[试验例4]根据处理美白物质叉头框蛋白O3a的核移动观察
对1X106个的MNT-1细胞(人黑色素瘤细胞系,龙沙集团,瑞士)分别使用预设的1nM、10nM、100nM、1μM、10μM或100μM的浓度的已知的美白物质维生素C、N-乙酰半胱氨酸及水溶性维生素E进行处理后,在37℃下培养48小时。作为对照组使用了非处理组。对培养的细胞分别用蓝色荧光(DAPI:核染色剂-蓝)、叉头框蛋白O3a IF抗体(红)(Cell SignalingTechnology公司),进行染色,并用荧光显微镜(EVOSfl digital fluorescencemicroscope,先进显微镜集团:Advanced Microscopy Group,美国AMG)进行观察,从而将其结果在图3中示出。
在图3的结果中,可以知道用维生素C、N-乙酰半胱氨酸及水溶性维生素E进行处理时,可以观察到叉头框蛋白O3a的核移动,在各情况下,在100μM中没有观察到核移动,相反,在100nM下可以观察到鲜明的核移动。
从所述试验例2至4的结果中可以确认,在观察到叉头框蛋白O3a核移动的物质浓度中示出美白功效,将对叉头框蛋白O3a的激活起到介导作用的物质为同时实现抗老化、抗氧化功效及美白功效的抗老化-美白物质。因此,可以知道通过本发明的筛选方法,能够筛选出试验物质的抗老化的同时客观正确地筛选抗氧化效果和美白效果。
Claims (6)
1.一种筛选具有美白功效的物质的方法,其特征在于,所述方法通过确认欲试验的物质是否激活叉头框蛋白O3a(Forkhead box O3A)或促进叉头框蛋白O3a(Forkhead boxO3A)的表达来确认所述物质是否为减少黑色素的生成从而显示美白功效的物质,所述筛选具有美白功效的物质的方法,其特征在于,所述确认包含以下步骤:
a)使用欲试验的物质对黑色素形成细胞进行处理;
b)观察在所述步骤a)中,经过试验物质处理后的黑色素形成细胞中的叉头框蛋白O3a的移动的步骤;以及
c)如果叉头框蛋白O3a在所述黑色素形成细胞中向核移动,则判定该物质为具有美白功效的物质。
2.根据权利要求1所述的筛选具有美白功效的物质的方法,其特征在于,通过所述筛选方法能够筛选出同时具有美白功效及抗老化功效的物质。
3.根据权利要求1所述的筛选具有美白功效的物质的方法,其特征在于,所述叉头框蛋白O3a的激活或促进叉头框蛋白O3a的表达是通过利用蛋白质印迹法(Western blot)、酶联免疫吸附测定(ELISA)或反转录酶-聚合酶链锁反应(RT-PCR)来确认的。
4.根据权利要求3所述的筛选具有美白功效的物质的方法,其特征在于,如果所述试验物质促进叉头框蛋白O3a的表达,则判定该物质为具有美白功效的物质。
5.根据权利要求3或4所述的筛选具有美白功效的物质的方法,其特征在于,通过所述筛选方法能够筛选出同时具有美白功效及抗老化功效的物质。
6.根据权利要求1所述的筛选具有美白功效的物质的方法,其特征在于,以1nM~100nM的浓度对所述欲试验的物质进行处理。
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