CN113667669A - 抑制酪氨酸酶表达的反义寡核苷酸及其应用 - Google Patents
抑制酪氨酸酶表达的反义寡核苷酸及其应用 Download PDFInfo
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Abstract
本发明公开一种抑制酪氨酸酶表达的反义寡核苷酸及其应用,属于生物医药领域。本发明利用RNA Structure v6.2设计了靶向人类酪氨酸酶(TYR)的反义寡核苷酸,筛选出能有效抑制TYR表达进而降低黑色素产生的反义寡核苷酸TYR‑2、TYR‑5。该反义寡核苷酸具有较高的递送效率和特异性靶向性;可以高效靶向人酪氨酸酶基因、进而降低黑色素合成,淡化色斑,避免了化学美白产品带来的有害化学物质损伤细胞、重金属沉积等副作用。为美白化妆品的研发提供了有效数据。
Description
技术领域
本发明属于生物医药领域,具体涉及一种抑制酪氨酸酶表达的反义寡核苷酸及其应用。
背景技术
随着生活水平的提升,消费者对美白产品的要求也日益提高,不论是产品的安全性还是美白效果都得到消费者的高度关注。这也对美白剂的研发提出了更高要求,因此选择高效安全提升美白效果的成分尤为重要。
黑色素是一种生物色素,是由人体内的黑色素细胞合成分泌。黑色素可吸收并消散99.9%紫外线[1],保护机体遭受紫外线的损害。黑色素的生成机理是:在酪氨酸酶的氧化催化下酪氨酸逐步形成多巴、多巴醌和多巴色素中间产物,最终形成皮肤黑素,导致皮肤的变黑[2]。在该合成途径中,酪氨酸酶具有关键性作用。因此可通过抑制酪氨酸酶基因的表达,来减少黑色素的合成。
反义寡核苷酸(antisense oligonucleotides,ASOs)是一类可与靶RNA通过碱基互补配对原理结合,并抑制其功能或诱导其降解的短链DNA或RNA分子。其中反义寡核苷酸片段长度一般由十至二十几个碱基组成。为了克服核酸酶的降解问题,反义寡核苷酸会进行一系列化学修饰,例如:硫代磷酸酯修饰[3]、2′-O-烷基修饰[4]等。基于已有的反义核酸化学修饰,为提高生物学效应和稳定性,有研究者合成了一种嵌合型的反义核酸,被称为“gap-mer”战略(gap-mer strategy)[5]。此外基于对细胞摄取ASOs机理的认识,针对在递送ASOs中存在的问题,人们主要从提高细胞摄取量、改变细胞摄取ASOs方式、增强ASOs从胞内体内释放等方面研究了大量的递送方法。例如:阳离子脂质体[6]、聚合纳米粒[7]和GalNAC技术[8]等。
在已有的文献报道中,常见抑制酪氨酸酶活性的美白成分,是酚类和酸类物质。但由于提取工艺繁琐、增白效果不明显、具有细胞毒性等不利因素,限制了在美白产品中的使用。针对抑制黑色素的合成,有研究者从抑制黑色素形成相关基因表达的角度出发。研发针对酪氨酸酶(TYR)、酪氨酸相关蛋白(TYRP)和小眼相关转录因子(Mitf)的小RNA美白产品[9]。随着反义寡核苷酸设计软件的更新和合成工艺的改良,其特异性随之增加,毒副作用也有所降低。因此,十分有必要开发基于反义寡核苷酸的美白产品。
参考文献
[1]MEREDITH P.RIESZJ.Radiative relaxation quantum yields forsyntheticeumelanin[J].Photochemistry&Photobiology,2010,79(2):211-216.
[2]吴长机,方一泓,张立萍.植物活性美白成分的研究进展[J].科技广场,2021,44(4):49-52.
[3]MilliganJF,MatteucciMD,MartinJC.Current concepts in anti-sensedrug design[J].Med Chem,1993,36(14):1923.
[4]LesnikEA,GuinossoCJ,KawasakiAM,etal.Oligonucleotides containing2′-O-modified adenosine:synthesis and effects on stability of DNA:RNAduplexes[J].Biochem,1993,32:7832.
[5]Altmann KH,DeanNM,FabbroD,etal.Second generation of antisenseoligonucleotides:from nuclease resistance to biological eff-macy in animais[J].Chimia,1996,50:171.
[6]Tari A M.Preparation and application of liposome-incorporated oligodeoxynucleotides.MethodsEnzymol,2000,313:372-388.
[7]Chavany C,Le Doan T,CouvreurP,et al.Polyalkylcyanoacrylatenanoparticles as polymeric carriers for antisense oligonculeotides[J].PharmRes,1992,9(4):441-449.
[8]Rosie Z.Yu,Rudy Gunawan,Noah Post,et al.Disposition andPharmacokineticsof a GalNAc3-Conjugated Antisense OligonucleotideTargetingHuman Lipoprotein(a)in Monkeys[J].Nucleic Acid Therapeutics.2016,26(6):372-380.
[9]黄兵,殷勤伟.一种小核酸美白祛斑霜及其制备方法:中国,CN201210369781.2[P],2012.
发明内容
为了克服现有技术的缺点与不足,本发明的目的在于提供一种抑制酪氨酸酶表达的反义寡核苷酸。
本发明的另一目的在于提供上述抑制酪氨酸酶表达的反义寡核苷酸的应用。
本发明的目的通过下述技术方案实现:
一种抑制酪氨酸酶表达的反义寡核苷酸,如TYR-2或TYR-5所示:
TYR-2:5′-AGGTCAGGCTTTTTGGCCCT-3′;
TYR-5:5′-TGGCTGCTTTTCTTCAGGAA-3′;
所述的抑制酪氨酸酶表达的反义寡核苷酸经过硫代磷酸化修饰,具体在5′端末尾连续3个碱基以上和3′端末尾连续3个碱基以上进行硫代磷酸化修饰。
上述抑制酪氨酸酶表达的反义寡核苷酸在制备用于抑制或祛除黑色素生成和沉积的化妆品组合物中的应用;
上述抑制酪氨酸酶表达的反义寡核苷酸在制备用于治疗黑色素相关疾病的药物中的应用。例如可将这种反义寡核苷酸作为药物给药于受药者身上特定部位,比如色素斑块。任选地,上述药物剂型中可以包含任何药学可接受的辅助剂,只要其适合于相应的给药体系、并且恰当地保持所述反义寡核苷酸的活性。
一种组合物,该组合物含有上述抑制酪氨酸酶表达的反义寡核苷酸;
进一步,所述组合物为药物组合物或化妆品组合物;所述药物组合物还含有任何药学可接受的辅助剂;所述化妆品组合物还含有常用的化妆品成分;
所述的化妆品组合物可以是乳剂、膏剂、霜剂、水剂、气雾剂或粉剂等。
本发明相对于现有技术具有如下的优点及效果:
(1)本发明利用RNA Structure v6.2设计了靶向人类酪氨酸酶(TYR)的反义寡核苷酸,筛选出能有效抑制TYR表达进而降低黑色素产生的反义寡核苷酸TYR-2、TYR-5。为美白化妆品的研发提供了有效数据。
(2)本发明的反义寡核苷酸具有较高的递送效率和特异性靶向性;可以高效靶向人酪氨酸酶(TYR)基因、进而降低黑色素合成,淡化色斑,避免了化学美白产品带来的有害化学物质损伤细胞、重金属沉积等副作用。
附图说明
图1是RNA structure v6.2软件设计TYR-2序列。
图2是RNA structure v6.2软件设计TYR-5序列。
图3是qPCR检测转染不同反义寡核苷酸后细胞中TYR mRNA表达水平的变化(**P<0.01,***P<0.001,结果表示为平均值±标准差;n=3)。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
下列实施例中未注明具体实验条件的试验方法,通常按照常规实验条件或按照制造厂所建议的实验条件。所使用的材料、试剂等,如无特殊说明,为从商业途径得到的试剂和材料。
实施例1
1材料与方法
1.1细胞与试剂
人类A375恶性黑色素瘤细胞由本实验室购买并保存。
DMEM培养基、胎牛血清、Opti-MEM培养基均购自美国GIBCO公司;Lipofectamine2000购自于Thermo Fisher/赛默飞世尔科技;Trizol购自于Invitrogen(美国)公司;2xSYBR Green qPCR Master Mix购自于Biosharp。
1.2引物合成
人酪氨酸酶TYR实时荧光定量PCR引物以及GADPH荧光定量引物均合成自生工生物工程(上海)股份有限公司,序列如表1所示。
表1荧光定量PCR引物
引物名称 | 序列(5′-3′) |
GAPDH-Forward | CAATGACCCCTTCATTGACC |
GAPDH-Reverse | GACAAGCTTCCCGTTCTCAG |
TYR-Forward | TGCACAGAGAGACGACTCTTG |
TYR-Reverse | GAGCTGATGGTATGCTTTGCTAA |
反义寡核苷酸的合成化学修饰:反义寡核苷酸的合成与硫代磷酸化修饰由生工生物工程(上海)股份有限公司完成反义寡核苷酸序列。序列如表2所示:
表2反义寡核苷酸序列
序列名称 | 序列(5′-3′) | 总自由能(Overall△G<sub>37</sub>) |
TYR-1 | TGTAGGATTCCCGGTTATGT | -21.4 |
TYR-2 | AGGTCAGGCTTTTTGGCCCT | -21.1 |
TYR-3 | CTGGATTTCTTGTTCCCACC | -20.9 |
TYR-4 | ACCAAATAGCATCCTTTCCT | -19.0 |
TYR-5 | TGGCTGCTTTTCTTCAGGAA | -19.1 |
SCR | CATTAATGTCGGACAACTCAAT | 0 |
2实验方法
2.1反义寡核苷酸的设计与合成
为设计高效靶向人类酪氨酸酶基因并抑制其功能的反义寡核苷酸,我们从NCBI(National Center for Biotechnology Information)中获取人类酪氨酸酶TYR mRNA全长序列。利用RNA structure v6.2软件,对TYR mRNA进行二级结构预测,之后在得到该二级结构的基础上,利用oligowalk模块设计靶向结合TYR mRNA的反义寡核苷酸片段。在oligowalk界面会显示反义寡核苷酸与TYR mRNA相结合的5项自由能数据,每移动一个碱基,界面又自动显示新的数据,其中总自由能(Overall△G37)是综合其他4项自由能指标得出的综合性指标,亦是筛选反义药物的依据。本文以TYR mRNA序列全长为靶点,设计反义寡核苷酸序列(见表2),并对每条序列的5′端末尾连续3个碱基和3′端末尾连续3个碱基进行硫代磷酸化骨架修饰。
随机寡核苷酸序列,记作SCR,其5′端末尾连续3个碱基和3′端末尾连续3个碱基也进行硫代磷酸化骨架修饰。
2.2A375恶性黑色素瘤细胞的传代培养
将人A375恶性黑色素瘤细胞(常规市售)培养于含体积分数为10%的胎牛血清,1%双抗的DMEM培养基中,置于37℃、体积分数为5%的CO2饱和湿度下培养箱进行培养。选用对数生长期的细胞进行实验,选用对数生长期的细胞进行实验。
细胞传代时,弃除原有培养基,用PBS缓冲液漂洗两次。加入1mL 0.25%胰酶-EDTA,将培养瓶放置于培养箱中消化2min左右,在显微镜下观察到细胞形态变圆时,加入完全培养基至瓶中终止消化,得到细胞悬液进行分瓶传代。
2.3A375细胞转染及分组处理
A375细胞分为SCR和ASO组。SCR组为转染经硫代磷酸化修饰的随机寡核苷酸序列(随机寡核苷酸SCR,见表2)的细胞,ASO组分别转染五条经硫代磷酸化修饰的预测反义寡核苷酸序列(反义寡核苷酸TYR-1~5,见表2)的细胞。SCR和ASO组的终浓度设置为0.5μmol/L。采用阳离子脂质体LipofectamineTM 2000转染,转染步骤如下:
(1)取对数生长期细胞,用有血清有抗生素的DMEM培养基调整细胞悬液浓度为3x105/mL,每孔1mL接种于6孔板。将培养板置于37℃,5%CO2,待细胞生长到60~70%时进行转染;
(2)在转染之前,吸除6孔板中的原培养基。加入1.5mL不含血清和双抗的DMEM培养基,进行饥饿处理;
(3)制备反义寡核苷酸-LipofectamineTM 2000混合液。
a.稀释转染试剂LipofectamineTM 2000;使用前,将LipofectamineTM 2000转染试剂轻轻摇匀,然后取5μL,用250μL不含血清的优化培养基(Opti-MEM)稀释,轻轻混合,室温孵育5min;
b.稀释反义寡核苷酸:用250μL不含血清的优化培养基(Opti-MEM)稀释10μL反义寡核苷酸;
c.稀释好的LipofectamineTM 2000经过5min的孵育后,将之与上述步骤b稀释好的反义寡核苷酸粒轻轻混合,室温孵育20min;
(4)将上述混合液加入含有细胞以及培养基的细胞培养板中,轻轻摇晃,使之混合;
(5)经过37℃培养6h后,将孔里含有脂质体混合液的培养基移去,更换成完全培养基(含血清、双抗);
(6)将培养板置于37℃,5%CO2,培养箱中培养48h后提取RNA。
2.4Real-time PCR检测反义寡核苷酸对TYR的降解能力
为检测不同反义寡核苷酸在被转染进入A375细胞后,对其靶基因TYR的降解能力,使用Real-time PCR方法检测转染经硫代磷酸化修饰的不同组反义寡核苷酸的细胞中TYRmRNA相对表达量,以此找到作用效果最佳的ASO,具体实验方法如下:
2.4.1细胞总RNA的提取
1)实验分组如下:SCR(随机组)和ASO组,每组3个复孔;
2)收集细胞,1500rpm,5min离心去掉上清;
3)各组加入1mL PBS转移至1.5mL EP管,1500rpm,5min,洗涤一遍;
4)各组分别加入1mL Trizol,充分混匀,冰上静置5min;
5)各组分别加入200μL氯仿,涡旋15s,冰上静置15min;
6)4℃,12000rpm,离心10min;
7)将上清(约400μL)转移至新的EP管,加入等体积异丙醇,-20℃静置10min;
8)4℃,12000rpm,离心10min;
9)去掉上清,加入75%乙醇洗涤RNA沉淀,4℃,12000rpm,重复洗涤三次;
10)去掉上清,将提取的总RNA于超净工作台上,自然风干;
11)加入适量的DEPC水,于-80℃保存。
2.4.2逆转录cDNA以及qPCR检测TYR-mRNA相对表达量
a.逆转录cDNA
1)使用NanoDrop 2000微量核酸定量仪测定所提取的RNA浓度;
2)根据逆转录试剂说明书以及RNA浓度定量结果,10μL逆转录体系按比例加入1μgRNA、逆转录试剂和无酶水至200μL EP管;
3)混合均匀后进行瞬时离心,放置于PCR仪中,按照以下程序完成进行逆转录;42℃15min,82℃2min,4℃保存。
b.qPCR检测TYR-mRNA相对表达量
1)根据2x SYBR Green qPCR Master Mix试剂说明书,按下列组份配制qPCR反应液(反应液配制需在冰上进行)
主要成分 | 每个反应用量(μL) |
2xSYBR Green qPCR Master Mix | 10 |
cDNA | 1 |
上游引物(10μM) | 1 |
下游引物(10μM) | 1 |
去离子水 | 补至20 |
总反应体系 | 20 |
设置三个复孔,并将上述组分轻柔混匀后加入八连管中,上机检测前进行瞬时离心。
2)扩增程序设置
两步法PCR扩增条件如下:Stage 1:预变性1个循环95℃30秒;Stage 2:qPCR反应40个循环95℃15秒,55℃30秒,72℃30秒;Stage 3:溶解曲线1个循环95℃15秒,60℃60秒,95℃15秒。
3)结果分析
以GAPDH作为内参,目的基因和内参Ct值用2-△△Ct计算目的基因的相对水平变化。
2.5黑色素含量的测定
1)选取处于生长对数期的A375恶性黑色素瘤细胞,以3x105/孔的数量铺种于6孔板中。
2)第二天待细胞融合度达到60%~70%时,利用脂质体LipofectamineTM 2000转染经硫代磷酸化修饰的反义寡核苷酸进A375细胞,细胞培养箱中培养72h。
3)经PBS缓冲液洗涤两次,每孔加入1mL 0.25%胰蛋白酶,在培养箱中放置2min左右进行消化,再加1mL完全培养基终止消化。
4)收集细胞悬液后1000r/min离心5min,弃除上清液。每孔加入1mL PBS缓冲液,将细胞吹散,血球计数板计数。
5)1000r/min离心5min,弃除上清液,样品经空气干燥,每106个细胞溶于500mL1mol/L含1%DMSO的NaOH溶液。
6)经加热80℃至1h后冷却,选择475nm波长在分光光度计上读取吸光度值A。黑色素含量相对抑制率%=(随机组吸光度值-实验组吸光度值)/随机组吸光度值%。
2.6统计学处理
数据均以至少三个生物学重复的平均值±SD表示,使用GraphPad Prism软件8.0版(SystatSoftware,SanJose,CA,USA)进行统计分析。使用不配对t检验(Student’s two-tailed unpaired t test),来分析各组数据间差异的显著性,并且p值<0.05被认为具有统计学意义。
3实验结果
3.1反义寡核苷酸的设计
反义寡核苷酸设计的关键在于对靶序列二级结构的准确模拟,以及设计的反义寡核苷酸对靶序列不同结合位点的覆盖度。本研究选取人酪氨酸酶TYR mRNA(NM_000372.5)全部序列,共2062个碱基。使用RNA structure v6.2对该序列进行二级结构预测。
随后通过OligoWalk功能对该二级结构中所有可能被反义寡核苷酸结合的位点都进行了分析,部分设计结果图1和图2所示。在该图界面,每移动一个碱基位点,程序会以该位点作为起点,计算反义寡核苷酸片段的吉布斯自由能和退火温度等参数。通过最小吉布斯自由能原理,经合理设计的反义寡核苷酸与靶序列的结合将倾向于使得热力学平衡条件下体系自由能更低。所以我们选择了吉布斯自由能相对较低的反义寡核苷酸作为预测序列,再通过实验进行验证。经过筛选,共有5条不同的潜在功能性的序列被设计出来。共设计5条TYR反义寡核苷酸序列,从TYR-1~TYR-5分别给予编号。这5条反义寡核苷酸序列所对应TYR靶点分别位于外显子exon4、exon5、exon1、exon1和exon5。
3.2不同反义寡核苷酸对TYR mRNA降解能力的检测
为了验证我们设计的反义寡核苷酸是否会抑制TYR mRNA的表达,使用lipo2000将经硫代磷酸化修饰的不同组反义寡核苷酸(0.5μM)转染进A375恶性黑色素瘤细胞中,培养48h后提取细胞的总RNA。采用实时荧光定量PCR对不同组别细胞中TYR mRNA的相对表达量进行检测。结果如图3所示,反义寡核苷酸TYR-1、TYR-2和TYR-5对TYR mRNA表达有着不同明显抑制程度。其中,TYR-2(P<0.001)和TYR-5(P<0.001)的抑制效果较好,均为极显著差异,TYR-1(P<0.01)的抑制效果次之,为显著差异。因为TYR-2和TYR-5抑制TYR的表达较为明显,故被筛选出来,作为有效序列,为后续的实验研究对象。TYR-2、TYR-5序列见表2。
3.3NaOH裂解法检测黑色素含量
为了进一步证明上述有效序列对酪氨酸酶基因表达的抑制,我们通过NaOH裂解法测定A375细胞中的黑色素含量。结果如表3所示,转染有经硫代磷酸化修饰的反义寡核苷酸TYR-2(0.5μM)与TYR-5(0.5μM)组别的黑色素含量与随机组(0.5μM)比较,有显著下调的作用,下调百分率分别是14%(P<0.05)和15%(P<0.05),均具有统计学差异。因此,反义寡核苷酸TYR-2和TYR-5都具有抑制TYR mRNA表达,进而降低黑色素合成的作用。
表3 NaOH裂解法检测A375细胞中的黑色素含量
分组 | A375 | 下调百分率 |
空白对照 | 0.212±0.003 | |
SCR | 0.205±0.007 | 0 |
TYR-2 | 0.177±0.004* | 14% |
TYR-5 | 0.175±0.005* | 15% |
注:与SCR组比较,*p<0.05;空白对照是指未转染寡核苷酸的A375细胞组。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 暨南大学
<120> 抑制酪氨酸酶表达的反义寡核苷酸及其应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> GAPDH-Forward
<400> 1
caatgacccc ttcattgacc 20
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> GAPDH-Reverse
<400> 2
gacaagcttc ccgttctcag 20
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TYR-Forward
<400> 3
tgcacagaga gacgactctt g 21
<210> 4
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TYR-Reverse
<400> 4
gagctgatgg tatgctttgc taa 23
<210> 5
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TYR-1
<400> 5
tgtaggattc ccggttatgt 20
<210> 6
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TYR-2
<400> 6
aggtcaggct ttttggccct 20
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TYR-3
<400> 7
ctggatttct tgttcccacc 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TYR-4
<400> 8
accaaatagc atcctttcct 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TYR-5
<400> 9
tggctgcttt tcttcaggaa 20
<210> 10
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> SCR
<400> 10
cattaatgtc ggacaactca at 22
Claims (10)
1.一种抑制酪氨酸酶表达的反义寡核苷酸,其特征在于:所述的反义寡核苷酸如TYR-2或TYR-5所示:
TYR-2:5′-AGGTCAGGCTTTTTGGCCCT-3′;
TYR-5:5′-TGGCTGCTTTTCTTCAGGAA-3′。
2.根据权利要求1所述的抑制酪氨酸酶表达的反义寡核苷酸,其特征在于:
所述的抑制酪氨酸酶表达的反义寡核苷酸经过硫代磷酸化修饰。
3.根据权利要求2所述的抑制酪氨酸酶表达的反义寡核苷酸,其特征在于:
所述的抑制酪氨酸酶表达的反义寡核苷酸在5′端末尾连续3个碱基以上和3′端末尾连续3个碱基以上进行硫代磷酸化修饰。
4.权利要求1~3任一项所述的抑制酪氨酸酶表达的反义寡核苷酸在制备用于抑制或祛除黑色素生成和沉积的化妆品组合物中的应用。
5.权利要求1~3任一项所述的抑制酪氨酸酶表达的反义寡核苷酸在制备用于治疗黑色素相关疾病的药物中的应用。
6.根据权利要求5所述的应用,其特征在于:直接给药于受药者身上的色素斑块。
7.一种组合物,其特征在于:所述组合物含有权利要求1~3任一项所述的抑制酪氨酸酶表达的反义寡核苷酸。
8.根据权利要求7所述的组合物,其特征在于:
所述组合物为药物组合物或化妆品组合物。
9.根据权利要求8所述的组合物,其特征在于:
所述药物组合物还含有任何药学可接受的辅助剂;
所述化妆品组合物还含有常用的化妆品成分。
10.根据权利要求9所述的组合物,其特征在于:
所述的化妆品组合物为乳剂、膏剂、霜剂、水剂、气雾剂或粉剂。
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