敲降TYRP1基因对香猪表皮黑素细胞黑色素生成的影响

doi:10.11843/j.issn.0366-6964.2023.10.013

摘要/Abstract

摘要： 旨在探究酪氨酸相关蛋白酶1（tyrosinase related protein 1，TYRP1）对香猪原代表皮黑素细胞黑色素生成的影响。本研究选用具有"两头乌"毛色特征的3月龄健康香猪3头，每头猪采取头部黑色皮肤组织和背部白色皮肤组织。通过HE染色观察不同颜色香猪皮肤的毛囊结构及黑素细胞的分布特征；体外培养香猪原代表皮黑素细胞并通过多巴（L-Dopa）染色、实时荧光定量PCR和Western Blot方法进行鉴定；根据TYRP1基因序列构建5条siRNA，采用实时荧光定量PCR筛选出干扰效率最高的siRNA进行转染。成功干扰黑素细胞内TYRP1的表达后，分别通过实时荧光定量PCR、Western Blot、黑色素含量检测方法对黑素细胞内TYR、TYRP1及TYRP2的mRNA与蛋白相对表达量、细胞内总黑色素含量进行检测。结果显示，在香猪黑色和白色皮肤中都观察到完整的毛囊结构，黑素细胞主要分布在香猪黑色被毛皮肤中的毛囊外根鞘部位以及表皮。在MelM培养基作用下，黑素细胞生长旺盛，细胞形态、生长曲线、多巴染色、实时荧光定量PCR及Western Blot结果均表明培养的黑素细胞维持正常的生物学特性。敲降TYRP1后下调香猪表皮黑素细胞中黑色素生成相关基因TYR、TYRP1和TYRP2的mRNA和蛋白的表达，同时对黑色素的形成具有抑制作用。TYRP1基因能够影响香猪表皮黑素细胞黑色素的生成，研究结果可为探索TYRP1基因对香猪黑色素沉积的分子机制提供试验参考和基础数据。

关键词: TYRP1, 黑素细胞, siRNA, HE染色, 多巴染色, 黑色素

Abstract: The aim of this study was to explore the effect of tyrosine-related protease 1 (tyrosinase related protein 1, TYRP1) on melanin production in skin melanocytes of Xiang pigs. Three 3-month-old healthy pigs with the hair color characteristics of "two-head-black" animals were selected. The black skin tissue on the head and white skin tissue on the back were collected for each pig.The structure of hair follicles and the distribution characteristics of melanocytes were observed by HE staining; The epidermal melanocytes were cultured in vitro and identified by Dopa (L-Dopa) staining, quantitative real-time PCR and Western Blot methods; Five siRNAs were constructed from TYRP1 gene sequence, and the siRNA with the highest interference efficiency was transfected by quantitative real-time PCR. After successful interference of TYRP1 in melanocytes, the mRNA and protein expression levels of TYRP, TYRP1 and TYRP2 were measured by quantitative real-time PCR and Western Blot, the total intracellular melanin content also been measured. The results showed that intact hair follicle structures were observed in both the black and white skin of Xiang pigs, and the melanocytes were mainly distributed in the outer root sheath of hair follicles and epidermes in the black hairy skin of Xiang pig. Under the treatment of MelM medium, the melanocytes grew vigorously, and the results of cell morphology, growth curve, Dopa staining, quantitative real-time PCR and Western Blot all indicated that the cultured melanocytes maintained normal biological characteristics. Knocking down of TYRP1 downregulated the mRNA and protein expression of melanogenesis-related genes TYR, TYRP1 and TYRP2 in Xiang pig epidermal melanocytes, and also had an inhibitory effect on melanin formation. The TYRP1 gene was able to affect the melanin production in melanocyte epidermal cells,the results provide experimental reference and basic data to explore the molecular mechanism of TYRP1 gene on melanin deposition in Xiang pigs.

Key words: TYRP1 gene, melanocytes, siRNA, HE staining, L-Dopa staining, melanin

中图分类号:

S828.2

毕欢, 覃海, 袁巍, 张雨丹, 张依裕, 陈伟. 敲降TYRP1基因对香猪表皮黑素细胞黑色素生成的影响[J]. 畜牧兽医学报, 2023, 54(10): 4143-4153.

BI Huan, QIN Hai, YUAN Wei, ZHANG Yudan, ZHANG Yiyu, CHEN Wei. Effect of Knocking down TYRP1 Gene on Melanin Production in the Xiang Pig Epidermal Melanocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4143-4153.

参考文献 36

[1]	胡今尧.猪的遗传及育种[M].长沙:湖南科学技术出版社, 1982:114-119.HU J Y.Genetic breeding of pigs[M].Changsha:Hunan Science and Technology Publishing House, 1982:114-119.(in Chinese)
[2]	LAMOREUX M L, WAKAMATSU K, ITO S.Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin[J].Pigment Cell Res, 2001, 14(1):23-31.
[3]	刘建, 李静如, 朱吉, 等.湘村黑猪毛色遗传的探讨[J].养猪, 2012(3):60-61.LIU J, LI J R, ZHU J, et al.Discussion on the inheritance of hair color in Xiangcun black pigs[J].Pig Farming, 2012(3):60-61.(in Chinese)
[4]	LE L, SIRÉS-CAMPOS J, RAPOSO G, et al.Melanosome biogenesis in the pigmentation of mammalian skin[J].Integr Comp Biol, 2021, 61(4):1517-1545.
[5]	白小青.荣昌猪毛色分子遗传标记的研究[D].咸阳:西北农林科技大学, 2004.BAI X Q.Molecular genetic markers of hair color in Rongchang pigs[D].Xianyang:Northwest A & F University, 2004.(in Chinese)
[6]	WANG W, DI T M, WANG W W, et al.EGCG, GCG, TFDG, or TSA inhibiting melanin synthesis by downregulating MC1R expression[J].Int J Mol Sci, 2023, 24(13):11017.
[7]	PALACZ K, CHOLEWA M, BONAR M, et al.The rate and quality of post-mortem hair root changes in relation to melanin content[J].Forensic Sci Int, 2023, 350:111784.
[8]	ADAMEYKO I, LALLEMEND F, AQUINO J B, et al.Schwann cell precursors from nerve innervation are a cellular origin of melanocytes in skin[J].Cell, 2009, 139(2):366-379.
[9]	OSUNA I, DOLINSKA M B, SERGEEV Y V.In vitro reconstitution of the melanin pathway's catalytic activities using tyrosinase nanoparticles[J].Int J Mol Sci, 2023, 24(1):639.
[10]	MAKPOL D S, JAM F A, RAHIM N A, et al.Comparable down-regulation of TYR, TYRP1 and TYRP2 genes and inhibition of melanogenesis by tyrostat, tocotrienol-rich fraction and tocopherol in human skin melanocytes improves skin pigmentation[J]. Clin Ter, 2014, 165(1):e39-e45.
[11]	CARO T.Bear essentials[J].Trends Genet, 2023, 39(4):233-234.
[12]	DELL'ANGELICA E C.Melanosome biogenesis:shedding light on the origin of an obscure organelle[J].Trends Cell Biol, 2003, 13(10):503-506.
[13]	MANGA P, KROMBERG J G R, BOX N F, et al.Rufous oculocutaneous albinism in southern African Blacks is caused by mutations in the TYRP1 gene[J].Am J Hum Genet, 1997, 61(5):1095-1101.
[14]	CARGILL E J, FAMULA T R, SCHNABEL R D, et al.The color of a Dalmatian's spots:linkage evidence to support the TYRP1 gene[J].BMC Vet Res, 2005, 1:1.
[15]	覃海, 陈伟, 毕欢, 等.基于转录组测序对剑白香猪黑色素生成相关基因的筛选与分析[J].中国兽医学报, 2022, 42(11):2261-2271, 2283.QIN H, CHEN W, BI H, et al.Screening and analysis of genes related to melanin production in Jianbaixiang pig based on transcriptome sequencing[J].Chinese Journal of Veterinary Science, 2022, 42(11):2261-2271, 2283.(in Chinese)
[16]	谢光跃, 姜勋平, 刘桂琼.黑色素合成的细胞基础及影响因素[J].畜牧与兽医, 2014, 46(1):109-112.XIE G Y, JIANG X P, LIU G Q.Cellular basis and influencing factors of melanin synthesis[J].Animal Husbandry & Veterinary Medicine, 2014, 46(1):109-112.(in Chinese)
[17]	高泽成, 吴晓云, 梁春年, 等.两种毛色牦牛皮肤黑色素组织学分析[J].四川农业大学学报, 2017, 35(1):110-115.GAO Z C, WU X Y, LIANG C N, et al.Melanin histologic analysis of yak skin in two different colors[J].Journal of Sichuan Agricultural University, 2017, 35(1):110-115.(in Chinese)
[18]	谢光跃, 刘桂琼, 姜勋平, 等.乌骨山羊皮肤黑色素细胞的分布及特征[J].华中农业大学学报, 2014, 33(2):83-88.XIE G Y, LIU G Q, JIANG X P, et al.Location and characteristics of melanocytes of black-boned goat skin[J].Journal of Huazhong Agricultural University, 2014, 33(2):83-88.(in Chinese)
[19]	张建, 陈伟, 王慧, 等.猪毛色遗传机制的研究进展[J].猪业科学, 2013, 30(1):100-103.ZHANG J, CHEN W, WANG H, et al.Progress in studying the genetic mechanism of hair color in pigs[J].Pig Industry Science, 2013, 30(1):100-103.(in Chinese)
[20]	白瑞, 于志慧, 范瑞文, 等.羊驼皮肤黑素细胞体外培养鉴定[J].畜牧兽医学报, 2013, 44(4):549-556.BAI R, YU Z H, FAN R W, et al.Culture, identification of alpaca skin melanocytes in vitro[J].Acta Veterinaria et Zootechnica Sinica, 2013, 44(4):549-556.(in Chinese)
[21]	姬凯元, 范瑞文, 张俊珍, 等.小鼠正常黑色素细胞与黑色素瘤细胞(B16)mRNA表达差异分析[J].中国生物化学与分子生物学报, 2019, 35(1):92-100.JI K Y, FAN R W, ZHANG J Z, et al.Differential expression of mRNA between murine melanocytes and B16 melanoma cells[J].China Journal of Biochemistry and Molecular Biology, 2019, 35(1):92-100.(in Chinese)
[22]	熊渺, 黄娟, 许兰娇, 等.泰和乌骨鸡鸡胚皮肤黑色素细胞的分离与培养[J].中国兽医学报, 2015, 35(12):1984-1990.XIONG M, HUANG J, XU L J, et al.Isolation and cultivation of melanocytes from embryo skin of Taihe silky fowl[J].Chinese Journal of Veterinary Science, 2015, 35(12):1984-1990.(in Chinese)
[23]	OSUNA I, DOLINSKA M B, SERGEEV Y V.In vitro reconstitution of the melanin pathway's catalytic activities using tyrosinase nanoparticles[J].Int J Mol Sci, 2023, 24(1):639.
[24]	PATEL M H, DOLINSKA M B, SERGEEV Y V.Tyrp1 mutant variants associated with OCA3:computational characterization of protein stability and ligand binding[J].Int J Mol Sci, 2021, 22(19):10203.
[25]	DOLINSKA M B, ANDERSON D E, SERGEEV Y V.In vitro characterization of the intramelanosomal domain of human recombinant TYRP1 and its oculocutaneous albinism type 3-related mutant variants[J].Protein Sci, 2023, 32(1):e4518.
[26]	WAGATSUMA T, SUZUKI E, SHIOTSU M, et al.Pigmentation and TYRP1 expression are mediated by zinc through the early secretory pathway-resident ZNT proteins[J].Commun Biol, 2023, 6(1):403.
[27]	YUAN W, QIN H, BI H, et al.Ssc-mir-221-3p regulates melanin production in Xiang pigs melanocytes by targeting the TYRP1 gene[J].BMC Genomics, 2023, 24(1):369.
[28]	LAI X L, WICHERS H J, SOLER-LOPEZ M, et al.Structure of human tyrosinase related protein 1 reveals a binuclear zinc active site important for melanogenesis[J].Angew Chem Int Ed, 2017, 56(33):9812-9815.
[29]	LABERGE G S, DUVALL E, GRASMICK Z, et al.Recent advances in studies of skin color and skin cancer[J].Yale J Biol Med, 2020, 93(1):69-80.
[30]	WU X Q, ZHANG Y, SHEN L Y, et al.A 6-bp deletion in exon 8 and two mutations in introns of TYRP1 are associated with blond coat color in Liangshan pigs[J].Gene, 2016, 578(1):132-136.
[31]	REN J, MAO H, ZHANG Z, et al.A 6-bp deletion in the TYRP1 gene causes the brown colouration phenotype in Chinese indigenous pigs[J].Heredity, 2011, 106(5):862-868.
[32]	XU Q, LIU X M, CHAO Z, et al.Transcriptomic analysis of coding genes and non-coding RNAs reveals complex regulatory networks underlying the black back and white belly coat phenotype in Chinese Wuzhishan pigs[J].Genes, 2019, 10(3):201.
[33]	CHIU Y L, RANA T M.siRNA function in RNAi:a chemical modification analysis[J].RNA, 2003, 9(9):1034-1048.
[34]	OZCAN G, OZPOLAT B, COLEMAN R L, et al.Preclinical and clinical development of siRNA-based therapeutics[J].Adv Drug Deliv Rev, 2015, 87:108-119.
[35]	LI G, ZHU X R, LIU C.Characterization of immune infiltration and construction of a prediction model for overall survival in melanoma patients[J].Front Oncol, 2021, 11:639059.
[36]	GILOT D, MIGAULT M, BACHELOT L, et al.A non-coding function of TYRP1 mRNA promotes melanoma growth[J].Nat Cell Biol, 2017, 19(11):1348-1357.