CN116356019A - 与人黑色素瘤相关的分子标记物krtdap及其应用 - Google Patents
与人黑色素瘤相关的分子标记物krtdap及其应用 Download PDFInfo
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Abstract
本发明属于黑色素瘤生物诊断技术领域,具体公开了一种与人黑色素瘤相关的分子标记物KRTDAP及其应用。所述分子标记物KRTDAP的氨基酸序列如SEQ NO.1所示,扩增所述分子标记物KRTDAP的引物为KRTDAP‑F:5’‑CTTTAACACCCCGTTCCTGA‑3’KRTDAP‑R:5’‑CTTCCAGTGGAGGTCATGGT‑3’。通过研究表明,keratinocyte differentiation associated protein(KRTDAP)表达水平降低可以作为诊断黑色素瘤发生及预后的生物标记物,其抗体可用于黑色素瘤诊断试剂盒的使用。
Description
技术领域
本发明属于分析化学及临床医学领域,具体涉及一种与人黑色素瘤相关的分子标记物KRTDAP及其应用。
背景技术
黑色素瘤(melanoma)是人类皮肤癌中恶性程度极高的肿瘤。近年来,由于环境改变、日光浴等因素,黑色素瘤在国内的发病率以每年3%-5%的速度逐年增长。与其他皮肤癌相比,黑色素瘤进展迅速,病情险恶,预后极差,导致了80%的皮肤肿瘤患者死亡,而早发现可明显提高患者生存率。因此,早发现、早诊断、早治疗是改善恶性黑色素瘤预后的关键。
目前皮肤恶性黑色素瘤尚无特异性肿瘤标志物,大部分常规实验室检查仅用于术前评估。由于黑色素瘤的形态多变,免疫组化在黑色素瘤的诊断与鉴别诊断中起着重要作用。而相关基因表达水平的检测也是诊断黑色素瘤的重要手段,但目前临床上尚未常规使用。因此,旨在开发新的分子标记物,提高黑色素瘤患者的早期诊断率,从而提升黑色素瘤患者的生存率。
发明内容
针对现有技术的不足,本发明提供了一种与人黑色素瘤相关的分子标记物KRTDAP及其应用,通过研究证明在人黑色素瘤组织中KRTDAP蛋白的表达明显低于正常皮肤组织,且与不良预后相关,即keratinocyte differentiation associated protein(KRTDAP)表达水平降低可以作为诊断黑色素瘤发生及预后的生物标记物,其抗体可用于制备黑色素瘤诊断试剂盒。
为解决现有技术问题,本发明采取的技术方案为:
与人黑色素瘤相关的分子标记物KRTDAP在制备黑色素瘤诊断、预后评估、或治疗及辅助治疗的相关产品上的应用,所述分子标记物KRTDAP的氨基酸序列如SEQ NO.1所示。
SEQ NO.1:
MKIPVLPAVVLLSLLVLHSAQGATLGGPEEESTIENYASRPEAFNTPFLNIDKLRSAFKADEFLN WHALFESIKRKLPFLNWDAFPKLKGLRSATPDAQ
作为改进的是,编码所述分子标记物KRTDAP的核苷酸序列如SEQ NO.2所示。
SEQ NO.2:
AGAGCACCCCAAACTTGACGCCATGAAGATCCCGGTCCTTCCTGCCGTGGTGCTCCTCTCCCTCC
TGGTG
CTCCACTCTGCCCAGGGAGCCACCCTGGGTGGTCCTGAGGTGAGCACGTCACCTGCATTTTTATT
CTTAT
ACGTGTTACCCACCTACCCCTCCATCTCTCTACCTCCTGTCCATTCCTGGATAAGTGGCTCTTAG
TTTTG
GATTCTGATGGGGGCTGAAGAAACTGGGGCTCTGAGGAAGGGAGGAGGGAACAGAGCATCCCTGG
GGAGA
CATTTGGGACAGAGATGCTCCTCTTTCTGGAAGGGATGGTGAAGTGTGTTGGGGAGGCTCGTGTA
CTGAA
CCTTGGGACTGAGTGGGTGGTCTGGGTTCAGACTATCTCCACCATGGGCTCTCAAGAGTGTTCAC
CCATT
CCGTTCCAGCTGCACACATGCCTGGGGACGTGCAGGGGAAGAGGGGGAACTGGAGAACGGCAATG
TGAGG
CAGAGCAGCTCCAAACCCAGGCCTGCATCCACATGGGCAGCAGGTGGAGAGGGTGAAGCAAAGCA
AAAAA
CTAGCTAGTTCTGCCTGCAGCTGACTTAACTCAAAGAGGAGTGGGGCTGGGGTGTTCTGGAGACA
CCCCT
GCTTAGCTCCTCTTTGCTTCATTTGTGCCCCTGGGAGTGGTGCCCAGGTGACGGGTGGCTCCCTG
CTGTC
TCTGGAGCATAGGCCACAGTGAGAATCTTTACCATCCAGTCCTCAGCCAGAGAGGAATCCTGATG
CCTAT
TCCGGATTTAAAAGTCTAAGACAGGGGCACAGGCTCAGCTGCTAAGGACCAAGCCAGTGGTGTCA
CTGAG
CTGGGGGGGCTGTCAGGGCCTGAGGTGAAATGGGGCCAACTTTCCCAGTCTGAAAGGGCAGCTTT
CACCC
CACCTGACGCCATGGATTGGGAAGATGTGCCTAGAGTTTTACATTTTTCAGGGGAAACTAGAAAT
CTGGA
TGAATTTATGAATTCTTACTAATTCAAAATGTCTAATTCAAACCAAAAAACAAATGACCAAATTT
TTATG
AGATCTAAGCATCTGGATCCAGCCTGCGGGCTCCGTGTTGGGCTCTGTCATGGGGAAGACACGAT
GGGAC
ACACTGAGGCCTGGGGTGGGGTCCCCATGGAGGGGGATTATGGAACCTCCAACAGTGGTGGCAAT
AAATC
CACAAAGCAAAGCAACCTCGATCAGTGAGAGGCAGGCGGGTCACCTCAGCAGCTGGGCTCCCACC
TGCTC
GCAAATCTTTCCCATCCAGCTATACGGACTGAGCGTGGGTGTCTCCAGAACACCCCACCTCTTAG
ACTTA
AGTCAACTGCAGGACAGCACTAGCTAGCTTTTCCGGAGAGGAATGCTCGGCTCCAGGCTTTCTGC
CTGCT
GCCCATGGTGACACTGCTTACATGTACCATTCTCCAATTCCCCCTCCTCACCCTGCACATCCGCC
AACCA
TGTGCGCGACTGGAACGGAGACCATGGTGGGGAATGGGTTGCGTTCTTTCGGGGACTGGGCTGTG
GATGA
AGGCAGAGCTGGCAGACAGGGATGTGGGCAAGGGCCCTGGCATCACGTGACCCCCTGCCGCCTTT
TCTCC
CTCTCCTTTTCCAGGAAGAAAGCACCATTGAGAATTATGCGTCACGACCCGAGGTAAGCCGTCTG
TTTTC
CCCGGCCCTCCAGCCACGAGTCTGTCCTCTCTCTCTTTTTGCTTCTTAGCAACTTTCCTAACACC
CTGCG
TTTCTGCCTAGGCCTTTAACACCCCGTTCCTGAACATCGACAAATTGCGATCTGTGAGTACGCTT
CTCTG
GTGCTACCCCCACGAATACCCTCATCACAGAGGGGATAAACGCCTTGGTAGAGAGCTTGCAAAGC
TGCCA
CGGTTCCTTCACCTTCTCCAGAGGGCTGGGGGCTAAGCTCTGGCCTCCCCTGGAGCTGACCTGCT
TCTGC
TGTCTCTCTTCCTCCTCAGGCGTTTAAGGCTGATGAGTTCCTGAACTGGCACGCCCTCTTTGAGG
TGAGT
GCTCCGGCGCCCCTTGCCCTCCTCGGTTGTCTGCAGTCATCCCAGGGTAGTGGCCACACATTTTG
GCTTT
AAATGTATGGGTTGGTTCCTGATCTCTGGGAGGGAGTGACTGTCCTGCCTGGATGCTGCCCAATG
GGCTG
TCAACGGAAGAAAGCTTCCCCCGGCGCCAGTCCTGGCTACCCGTGTTTTCCTAAGATGGTGGTGG
AGGAG
CAGGGCCCCAAGGACAATTGCAGAAGAGGAAGAGGCTAATCGGTCAAGATTTTATTTGAATCCCC
TCATC
CTCTATGGAAAAACTGAGGCCCACCCGCACTTAGAAGGCAAAGTCAGCGACAGAGCTGGGGCCCG
CGTCT
CCTGACTCTGCACTGACTTCTCACATGGCCTCATGCGAACGCCTTCAGCTCCCTCGGCCTCCGCT
TCCTC
TGTGATGTGGGGATGATGACAATTCCCACCACAAAAGGTTGCTGTAAAAACCCACTGAAATCATG
CTTGT
AGAGCAGCCCGTGCAAGGCAGACACAGACTAAGGCCTCAGGAAATATTAGCTCTTGATAAAGTAA
TGACA
AAATAATGACATTATTGTTACTATGGTTACTGTGGGAGTTCATTAAGGGAATGCTTACCCATCCA
TGCCC
GGCTGACCTGCAACACATCACTCTCTTTCTCTTTCTTTCTTTTAGTCTATCAAAAGGAAACTTCC
TTTCC
TCAACTGGGATGCCTTTCCTAAGGTAAGAGGTGTGGGCATTAGGAGGGTATGGAGCTTGGGGGTA
AGGGA
AGAAGCAGAAGATATGGGAGAGAGAGTGGGGACAGGGAAAGGAGGATCAGCCTTCTCACCAAACT
GAAGG
GAGGTCTTAGCTACTACATATAGAGGAGAACGGGAACTCACACCGAGGCAGAAATTCCTTTCCAT
CCATG
CCATCCTATACTGGGCACCCACGGGTTCCCCATCTAACCCTGACCCCTCTCCCTCTAGCTGAAAG
GACTG
AGGAGCGCAACTCCTGATGCCCAGTGACCATGACCTCCACTGGAAGAGGGGGCTAGCGTGAGCGC
TGATT
CTCAACCTACCATAACTCTTTCCTGCCTCAGGAACTCCAATAAAACATTTTCCATCCAACA
作为改进的是,所述相关产品为检测试剂。
一种黑色素瘤诊断、预后评估、或治疗及辅助治疗的检测试剂,所述检测试剂含有上述的分子标记物KRTDAP。
进一步改进的是,所述检测试剂含有抗体,所述抗体特异性结合分子标记物KRTDAP,检测方法为免疫组织化学法。
进一步改进的是,所述抗体为单克隆抗体或者多克隆抗体。
进一步改进的是,所述检测试剂包含扩增标记物KRTDAP的引物为:
KRTDAP-F:5’-CTTTAACACCCCGTTCCTGA-3’;
KRTDAP-R:5’-CTTCCAGTGGAGGTCATGGT-3’;
所述检测试剂使用的检测方法为qRT-PCR法。
一种检测试剂盒,包含上述的检测试剂。
有益效果:
与现有技术相比,本发明一种与人黑色素瘤相关的分子标记物KRTDAP及其应用,具有如下优势:该分子标记物在黑色素瘤肿瘤组织的表达明显低于相应的正常癌旁组织,且其表达量与肿瘤的临床分期呈负相关,与预后、生存率呈正相关,可以作为黑色素瘤早期筛查,预后评估指标。
附图说明
图1为KRTDAP在不同器官中的表达情况;
图2为KRTDAP在男女之间的表达差异;
图3为KRTDAP在TIMER(version=2.0)中的泛癌情况;
图4为KRTDAP在TCGA数据库中正常组织、原发黑色素瘤组织和转移黑色素瘤组织之中的表达情况;
图5为KRTDAP高表达组和低表达组SKCM患者的Kaplan-Meier生存曲线(p<0.001);
图6为总体1年、3年和5年OS的ROC曲线;
图7为单因素Cox回归分析KRTDAP表达、年龄、性别、病理分期、T期、M期、N期。
图8为多因素Cox回归分析KRTDAP表达、年龄、性别、病理分期、T期、M期、N期;
图9为临床特征列线图(性别、年龄、病理分期、KRTDAP表达、T期、M期、N期);
图10为免疫组织化学染色法检测临床黑色素瘤患者肿瘤组织(melanoma)及其对应的正常癌旁组织中KRTDAP表达情况;
图11为临床黑色素瘤患者肿瘤组织(melanoma)及其对应的正常癌旁组织中KRTDAP免疫组织化学染色统计结果;
图12为Western Blot法检测临床黑色素瘤患者肿瘤组织(melanoma)及其对应的正常癌旁组织中KRTDAP蛋白表达情况;
图13为临床黑色素瘤患者肿瘤组织(melanoma)及其对应的正常癌旁组织中KRTDAP蛋白表达水平的统计结果;
图14为临床黑色素瘤患者肿瘤组织(melanoma)及其对应的正常癌旁组织中KRTDAP mRNA表达水平的统计结果。
具体实施方式
实施例1数据库分析
黑色素瘤患者的RNA测序数据及相应临床信息分别从癌症基因组图谱数据库(TCGA,n=472)和基因型组织表达(GTEx,n=323)项目下载相关临床信息。在TCGA数据库的样本中,有1个正常样本和471个癌症样本,所以从GTEx数据库中选择了323个正常样本进行分析,其中,根据TCGA提供的样本信息将黑色素瘤患者区分为原发型和转移型。通过分析了正常样本、原发样本和转移性样本中KRTDAP的表达差异。结果显示,三者之间均有显著性差异,KRTDAP的表达随着黑色素瘤疾病的进展而逐渐下调。
根据患者的临床资料以及KRTDAP基因的表达情况将患者分为两组,采用Ka plan-Meier法分析所有肿瘤样本的生存情况,并对结果进行显著性检验(p<0.001)。此外,通过采用受试者工作特征(ROC)曲线分析和单因素和多因素Cox回归分析来探讨其诊断和预后能力。
采用Cox比例风险模型和多元logistic回归分析选择特征,构建包含所选的预测因素:性别、年龄、克拉克分期、癌症M分期、癌症T分期、癌症N分期和KRTDAP的表达。使用校准图评估了模型的校准、鉴别和临床有效性。结果发现,KRTDAP的表达量显著影响了患者的生存时间,并可以作为一个独立指标去预测患者的生存预后情况。
实施例2临床样本分析
采用黑色素瘤临床样本均来自中国医学科学院皮肤病医院,均征求病人的同意并经中国医学科学院皮肤病医院伦理委员会批准。
(1)组织标本来源及收集
中国医学科学院皮肤病医院收集到36例临床黑色素瘤患者手术切除的组织样本。
组织样本分别为黑色素瘤组织样本与距离肿瘤组织5cm以上的正常皮肤组织,手术中组织离体后一部分立即放入4%多聚甲醛进行固定,后续用于切片和免疫组织化学染色;另一部分立即放入液氮中冷冻后转移储存在-80℃冰箱,用于后续RNA和蛋白质的提取。
(2)组织切片及免疫组化
a)组织切片在4%多聚甲醛固定24h;
b)固定好的组织切片放入包埋框包埋后进行脱水:梯度酒精(70%、80%、95%、95%、100%I和100%II)各45min,酒精-二甲苯30min,二甲苯20min,二甲苯-石蜡30min,低熔点石蜡45min,高熔点石蜡45min;
c)将组织切片进行石蜡包埋,利用切片机进行切片(5μm厚),将切好的组织片放在37℃烘箱过夜。
d)继续将石蜡切片放置55℃烘箱30min,然后依次进行二甲苯脱蜡,梯度酒精水化(70%、80%、95%、95%、100%I和100%II)各3min,流水冲洗5min;
e)切片置于柠檬酸盐缓冲液中,沸水水浴10min进行抗原修复,取出后自然冷却;
f)将冷却后的切片置于PBS中在摇床上洗三次,每次5min;
g)将3%过氧化氢溶液,滴在切片的组织上,37℃放置20min后,用PBS洗三次,每次5min;
h)将封闭血清(10%羊血清)滴加在切片上,37℃放置60min;
i)轻甩去血清,将0.5%BSA-PBS稀释的KRTDAP抗体(稀释比例1:200),滴加在切片上,4℃冰箱过夜;
j)第二天取出片切片于室温放置60min,PBS洗三次,每次5min;
k)将0.5%BSA-PBS稀释的HRP标记的羊抗兔二抗(稀释比例1:1000),滴加在切片上,37℃孵育60min,PBS洗三次,每次5min;
l)配制DAB显影液,并滴加在切片上,室温反应,镜下观察,根据显色情况,在水中终止反应,记录显色时长,流水冲洗5min;
m)苏木素染核1min,流水冲洗5min,镜下观察染核情况;
n)1%盐酸酒精中和2s,流水冲洗5min;
o)进行梯度酒精脱水:70%酒精、80%酒精、90%酒精、100%酒精I、100%酒精II各2min,二甲苯I、二甲苯II各2min;
p)中性树脂封片,通风橱晾干;并置于正置显微镜下拍照。
q)用Northern Eclipse软件进行图像定量分析,定量指标包括:阳性面积和阳性产物的总灰度值(Summary total gray,STG),其中,STG=阳性产物平均灰度值×阳性面积;阳性面积百分率=阳性面积÷检测视野面积。
利用免疫组织化学技术检测36对临床黑色素瘤组织及对应正常癌旁组织病理切片中KRTDAP的表达水平,发现KRTDAP在黑色素瘤组织中表达量明显降低。(3)Western Blot
a)组织总蛋白提取:对临床黑色素瘤组织及对应正常癌旁组织,加入含10%PI、10% PMSF的RIPA 200μl,匀浆后放在冰上静置5min,吹打并转移至EP管中,离心,4℃,12000rpm,30min;吸上清到新的EP管中,冰上暂存;按照碧云天BCA蛋白浓度测定试剂盒说明,测定蛋白浓度,用ddH2O将样本蛋白稀释到300μg/80μl,加入20μl 5*loading buffer,金属浴100℃,5min,室温冷却后-20℃冰箱储存。
b)配制10%分离胶与浓缩胶
试剂 | 分离胶(μl) | 浓缩胶(μl) |
ddH2O | 1900 | 1800 |
30%AA液 | 1700 | 300 |
1MTris-HCl(pH=8.8) | 1300 | \ |
1M Tris-HCl(pH=6.8) | \ | 750 |
10%SDS | 50 | 30 |
10%APS | 50 | 50 |
TEMED | 5 | 5 |
c)配制SDS-PAGE胶,先将配置好的分离胶灌入玻璃板,防治产生气泡,至距离玻璃板上沿1.5cm处,缓慢加入ddH2O压胶隔绝空气,静置30min左右胶面与水相有明显分界面,即分离胶凝固,倒出上层液体,配制上层浓缩胶,加入玻璃板后立即插上梳子,静置待凝固;
d)将凝胶板安装在电泳槽,在内外槽倒入1*电泳液,上样枪加样后,恒压85V开始电泳,直到marker分离,调整电压至110V,直至电泳完成;
e)电泳完成后,取出凝胶放在ddH2O中,将PVDF膜放在甲醇中激活20s,转到预冷的转膜液中;在转膜夹板中放好海绵及滤纸,再电流方向(从黑到红)放入凝胶和PVDF膜,凝胶在黑色板甲一侧,轻压排出每一层之间的气泡,合上夹板,放入转膜槽中,凝胶面(黑色一侧)朝向转膜槽负极,PVDF膜朝向正极,倒入预冷转膜液,放一块冰盒,将转膜槽放入4℃冰箱,保持低温转膜,恒流0.3A转膜90min;
f)转膜结束后,取出PVDF膜,ddH2O清洗两次,放入丽春红进行染色,观察是否有蛋白条带,判断转膜是否成功。清洗丽春红后,用5%牛奶-PBST进行封闭,室温1h;
g)封闭后的膜放入抗体孵育盒,加入按照1:10000比例用5%BSA/TBST稀释的KRTDAP一抗,4℃过夜;
h)第二天,用TBST洗膜,摇床上洗3次,每次5min,TBS洗一次,5min;加入1:10000稀释好的二抗,室温孵育1h;
i)TBST洗膜三次,每次5min,TBS洗一次,5min;
j)配制ECL显影液,将膜放置与化学发光成像系统进行曝光成像,拍照存档;
利用Western Blot技术检测临床黑色素瘤组织及对应正常癌旁组织中KRTDAP的表达水平,发现KRTDAP蛋白在黑色素瘤组织中表达量明显降低。
(4)Real-time PCR
a)组织总RNA提取:Trizol法提取组织总RNA。加入500μl Trizol。加入200ul氯仿,剧烈振摇,使上下两相充分混合,室温静置5min后,离心12000xg,15min 4℃离心。小心吸取上层水相160ul于新的RNase free EP管中,加入等体积异丙醇(160ul),颠倒混匀,室温放置10min后,12000xg,15min 4℃离心,离心后可见管内白色RNA沉淀。弃尽上清,使用预冷75%乙醇(DEPC水稀释)洗涤沉淀一次,7000g,5min 4℃离心。弃尽上清,室温放置晾干(10~15min),使得酒精挥发干净。之后加入15μl DEPC水溶解,测定浓度备用。
b)RNA逆转成cDNA:使用南京诺唯赞(vazyme)公司的逆转录试剂盒(去基因组DNA),按照试剂盒说明书进行RNA逆转录,逆转录体系如下:
试剂 | 用量 |
RNA | 1μg |
4×gDNA wiper Mix | 4μl |
RNase free ddH2O | 至16μl |
上述混合物混匀,PCR仪反应42℃2min,继续第二步逆转:
试剂 | 用量 |
上述混合物 | 16μl |
5×HiScriptⅡqRT SuperMixⅡ | 4μl |
混匀后,进行逆转录PCR,条件如下:50℃,15min;85℃,5s,得到的cD NA产物可用于Real-time PCR;
c)Real-time PCR:将上一步的cDNA作为模板进行Real-time PCR,使用诺唯赞(vazyme)公司的SYBR Green Mix,按照试剂盒说明书进行。10μl体系:
上述混合物混匀后,进行Real-time PCR,反应条件如下:95℃,5min;95℃,10s-60℃,30s;40个循环,
引物序列如下:
Primers | Sequence(5’-3’) |
β-actin-F | TCATGAAGTGTGACGTGGACAT |
β-actin-R | CTCAGGAGGAGCAATGATCTTG |
KRTDAP-F | CTTTAACACCCCGTTCCTGA |
KRTDAP-R | CTTCCAGTGGAGGTCATGGT |
利用Real-time PCR技术检测临床黑色素瘤组织及对应正常癌旁组织中KRTDAP的表达水平,发现KRTDAP mRNA的表达水平在黑色素瘤组织中显著降低。
综上所述,在黑色素瘤组织中KRTDAP的表达显著低于正常癌旁组织,证明KRTDAP可以作为黑色素瘤的分子标记物,并可应用于制备肿瘤的诊断试剂盒中可以作为黑色素瘤早期筛查,预后评估指标,具有良好的应用前景。
以上所述,仅为本发明较佳的具体实施方式,本发明的保护范围不限于此,任何熟悉本技术领域的技术人员在本发明披露的技术范围内,可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Claims (8)
1.与人黑色素瘤相关的分子标记物KRTDAP在制备黑色素瘤诊断、预后评估、或治疗及辅助治疗的相关产品上的应用,其特征在于,所述分子标记物KRTDAP的氨基酸序列如SEQNO.1所示。
2.根据权利要求1所述的应用,其特征在于,编码所述分子标记物KRTDAP的核苷酸序列如SEQ NO.2所示。
3.根据权利要求1所述的应用,其特征在于,所述相关产品为检测试剂。
4.一种黑色素瘤诊断、预后评估、或治疗及辅助治疗的检测试剂,其特征在于,所述检测试剂含有权利要求1所述的分子标记物KRTDAP。
5.根据权利要求4所述的检测试剂,其特征在于,所述检测试剂含有抗体,所述抗体特异性结合分子标记物KRTDAP,检测方法为免疫组织化学法。
6.根据权利要求5所述的检测试剂,其特征在于,所述抗体为单克隆抗体或者多克隆抗体。
7.根据权利要求6所述的检测试剂,其特征在于,所述检测试剂包含扩增标记物KRTDAP的引物为:
KRTDAP-F:5’- CTTTAACACCCCGTTCCTGA -3’;
KRTDAP-R:5’- CTTCCAGTGGAGGTCATGGT -3’;
所述检测试剂使用的检测方法为qRT-PCR法。
8.一种检测试剂盒,其特征在于,所述检测试剂盒包含权利要求4所述的检测试剂。
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