不同毛色牦牛皮肤组织学观察及MC1R基因功能验证

doi:10.11843/j.issn.0366-6964.2018.07.006

摘要/Abstract

摘要：

旨在阐明不同毛色牦牛皮肤组织形态学特点及MC1R调控黑色素合成的可能分子机制。采集大通牦牛（黑褐色）和天祝白牦牛（白色）各6头的背部皮肤组织，用甲苯胺蓝染色法分别对不同毛色牦牛皮肤组织进行染色，观察黑色素和成熟黑色素细胞的含量差异及分布特征，并通过实时荧光定量PCR（qRT-PCR）检测MC1R在不同毛色牦牛皮肤中的表达差异。B16小鼠黑色素瘤细胞中转染shRNA MC1R干扰载体后，应用qRT-PCR和Western blot检测转染效率，分析比较各组中与毛色相关基因（Agouti、MITF、TYR）的表达差异性，利用酶标仪检测不同时间点黑色素产量。结果表明，两种毛色牦牛的表皮和毛囊周围均分布着大量的黑色素细胞，但天祝白牦牛仅在表皮基底层和毛根处检测到少量黑色素，而大通牦牛表皮层和毛囊处产生大量黑色素。与大通牦牛相比，天祝白牦牛皮肤中MC1R表达量极显著降低（P<0.01）。在成功抑制MC1R的B16细胞中，TYR表达量极显著降低（P<0.01），MITF表达量显著降低（P<0.05），而Agouti表达量未发生显著变化（P>0.05）；同时黑色素含量减少。综上表明，黑色素大量沉积与MC1R高表达是导致牦牛毛色差异的原因。将shRNA MC1R载体成功转染B16小鼠黑色素瘤细胞后，MC1R的抑制情况与下游调控基因TYR表达一致，进而降低黑色素含量，说明MC1R对黑色素的合成及毛色形成起重要的调控作用。

Abstract:

The objectives of this study were to elucidate the characteristics of skin histomorphology in yaks with different coat colors and to discover the potential molecular mechanism by which MC1R induced melanin synthesis. Skin tissues were collected from the hindquarters of 6 Datong yaks(dark brown) and 6 Tianzhu yaks(white). Toluidine blue staining was used to reveal the content and distribution of both melanin and mature melanocytes in the skin tissues of different coat colors. MC1R expression was determined by quantitative real-time polymerase chain reaction(qRT-PCR). Afterward,shRNA interference vectors for MC1R were transfected into B16 murine melanoma cells,and transfection efficiency was quantified by qRT-PCR and Western blot assay. The expressions of hair color-related genes(Agouti,MITF and TYR) in each group were analyzed and compared,and melanin production was detected with a microplate reader at different time points. The results showed that a large number of melanocytes distributed around the epidermis and hair follicles of both types of yaks. Tiny amounts of melanin were detected at the base of the epidermis and the hair roots of Tianzhu white yak skin tissues,while large amounts of melanin were discovered in the epidermis and hair follicles of Datong yak skin tissues. The expression of MC1R in Tianzhu white yaks was significantly lower than that in Datong yaks(P<0.01).In B16 cells with successful inhibition of MC1R,expression of TYR was very significantly lower(P<0.01),MITF expression was significantly lower(P<0.05),Agouti expression was not significantly different(P>0.05),and melanin content was lower than in cells that expressed MC1R. These results indicate that MC1R plays an important role in melanin synthesis and formation of coat color. To summarize,shRNA MC1R vectors were successfully transfected into B16 mouse melanoma cells;inhibition of MC1R in B16 cells was consistent with reduced expression of the downstream gene TYR,which lowered the melanin content in skin tissues. Therefore,we conclude that massive deposition of melanin and high expression of MC1R are responsible for the differences in yak coat colors.

中图分类号:

S823.8⁺5.2

唐朋, 凌笑笑, 高泽成, 贾聪俊, 梁春年, 吴晓云, 褚敏, 阎萍. 不同毛色牦牛皮肤组织学观察及MC1R基因功能验证[J]. 畜牧兽医学报, 2018, 49(7): 1377-1386.

TANG Peng, LING Xiao-xiao, GAO Ze-cheng, JIA Cong-jun, LIANG Chun-nian, WU Xiao-yun, CHU Min, YAN Ping. Skin Histological Observation of Yak with Different Coat Colors and MC1R Functional Verification[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1377-1386.

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https://www.xmsyxb.com/CN/Y2018/V49/I7/1377

参考文献

[1] HUBBARD J K,UY J A C,HAUBER M E,et al.Vertebrate pigmentation:From underlying genes to adaptive function[J].Trends Genet,2010,26(5):231-239.
[2] RODRIGUES A R,ALMEIDA H,GOUVEIA A M.Intracellular signaling mechanisms of the melanocortin receptors:Current state of the art[J].Cell Mol Life Sci,2015,72(7):1331-1345.
[3] HADDADEEN C,LAI C,CHO S Y,et al.Variants of the melanocortin-1 receptor:Do they matter clinically?[J].Exp Dermatol,2015,24(1):5-9.
[4] 张俊珍,董常生,范瑞文,等.哺乳动物毛色形成研究进展[J].动物医学进展,2006,27(S1):65-68.
ZHANG J Z,DONG C S,FAN R W,et al.Advance in pigment melanin of mammalian[J].Progress in Veterinary Medicine,2006,27(S1):65-68. (in Chinese)
[5] STEINGRÍMSSON E,COPELAND N G,JENKINS N A.Melanocytes and the Microphthalmia transcription factor network[J].Annu Rev Genet,2004,38:365-411.
[6] 任玉红,杨刚,范瑞文,等.黑素皮质素受体1(MC1R)在不同毛色羊驼皮肤组织中的表达与定位研究[J].畜牧兽医学报,2012,43(7):1049-1055.
REN Y H,YANG G,FAN R W,et al.Expression and immunolocalization of melanocortin receptor 1(MC1R) in Alpaca skin with different coat color[J].Acta Veterinaria et Zootechnica Sinica,2012,43(7):1049-1055. (in Chinese)
[7] 李洪涛,曾献存,张文祥,等.哈萨克绵羊MC1R和ASIP基因多态性及表达量与被毛颜色表型相关性的研究[J].畜牧兽医学报,2013,44(3):366-375.
LI H T,ZENG X C,ZHANG W X,et al.Analysis of the relationship between polymorphism/mRNA expression levels of MC1R and ASIP and coat color phenotype in Kazakh sheep[J].Acta Veterinaria et Zootechnica Sinica,2013,44(3):366-375. (in Chinese)
[8] DONG Y J,WANG H D,CAO J,et al.Nitric oxide enhances melanogenesis of alpaca skin melanocytes in vitro by activating the MITF phosphorylation[J].Mol Cell Biochem,2011,352(1-2):255-260.
[9] 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)
[10] 赵彦斌,孙兆增,胡仲明,等.黑线仓鼠及其白化突变系毛囊中黑色素细胞的组织学分析[J].中国比较医学杂志,2010,20(10):1-2.
ZHAO Y B,SUN Z Z,HU Z M,et al.Histochemical study on the distribution of melanocytes in hair folicle of the Cricetulus barabensis and the albino mutant[J].Chinese Journal of Comparative Medicine,2010,20(10):1-2. (in Chinese)
[11] NISHIMURA E K.Melanocyte stem cells:A melanocyte reservoir in hair follicles for hair and skin pigmentation[J].Pigment Cell Melanoma Res,2011,24(3):401-410.
[12] COMMO S,GAILLARD O,BERNARD B A.Human hair greying is linked to a specific depletion of hair follicle melanocytes affecting both the bulb and the outer root sheath[J].Br J Dermatol,2004,150(3):435-443.
[13] 李鹏飞,董常生,杜海燕,等.黑色素皮质素受体1(MC1R)基因与哺乳动物毛色[J].畜牧兽医杂志,2006,25(1):21-22.
LI P F,DONG C S,DU H Y,et al.Melanocortin receptor 1(MC1R) and the hair color of mammalian[J].Journal of Animal Science and Veterinary Medicine,2006,25(1):21-22. (in Chinese)
[14] SLOMINSKI A,TOBIN D J,SHIBAHARA S,et al.Melanin pigmentation in mammalian skin and its hormonal regulation[J].Physiol Rev,2004,84(4):1155-1228.
[15] TOBIN D J,BYSTRYN J C.Different populations of melanocytes are present in hair follicles and epidermis[J].Pigment Cell Res,1996,9(6):304-310.
[16] 封竣淇,徐伟,黄兰,等.MC1R基因的研究进展[J].中国畜牧兽医,2017,44(4):1141-1148.
FENG J Q,XU W,HUANG L,et al.Research advances on MC1R gene[J].China Animal Husbandry & Veterinary Medicine,2017,44(4):1141-1148. (in Chinese)
[17] 杨翠军,葛剑,陈赛娟,等.6品系彩色獭兔MC1r和agouti基因mRNA表达量与毛色相关性[J].中国兽医学报,2016,36(8):1429-1434,1439.
YANG C J,GE J,CHEN S J,et al.Relationship between mRNA expression level of MC1r and agouti genes with coat color in 6 strains of color Rex rabbit[J].Chinese Journal of Veterinary Science,2016,36(8):1429-1434,1439. (in Chinese)
[18] 秦珂,黄翔,曾检华,等.陆川猪MC1R和KIT基因型鉴定[J].中国畜牧兽医,2017,44(3):773-781.
QIN K,HUANG X,ZENG J H,et al.Characterization of MC1R and KIT genotypes in Luchuan pig population[J].China Animal Husbandry & Veterinary Medicine,2017,44(3):773-781. (in Chinese)
[19] 王海东,李亚楠,陈天直,等.不同毛色绵羊皮肤中MC1R的差异表达及定位[J].基因组学与应用生物学,2016,35(10):2614-2620.
WANG H D,LI Y N,CHEN T Z,et al.Differential expression and localization analysis of MC1R in sheep skins with different coat color[J].Genomics and Applied Biology,2016,35(10):2614-2620. (in Chinese)
[20] TAIRA J,TSUCHIDA E,UEHARA M,et al.The leaf extract of Mallotus japonicus and its major active constituent,rutin,suppressed on melanin production in murine B16F1 melanoma[J].Asian Pac J Trop Biomed,2015,5(10):819-823.
[21] NINOMIYA K,MATSUMOTO T,CHAIPECH S,et al.Simultaneous quantitative analysis of 12 methoxyflavones with melanogenesis inhibitory activity from the rhizomes of Kaempferia parviflora[J].J Nat Med,2016,70(2):179-189.
[22] GRIMM D,STREETZ K L,JOPLING C L,et al.Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways[J].Nature,2006,441(7092):537-541.
[23] 孙笑尉,张雪莲,庞全海,等.RNAi对羊驼黑色素细胞中MC1R表达量及黑色素合成的影响[J].畜牧兽医学报,2015,46(7):1163-1168.
SUN X W,ZHANG X L,PANG Q H,et al.Effect of RNAi on MC1R expression and melanin synthesis in the melanocytes of alpaca[J].Acta Veterinaria et Zootechnica Sinica,2015,46(7):1163-1168. (in Chinese)
[24] 贾秀华.酪氨酸酶siRNA有效抑制小鼠酪氨酸酶基因表达的初步研究[D].广州:中山大学,2008.
JIA X H.The pilot study of tyrosinase siRNA effectively suppresses tyrosinase gene expression in mice[D].Guangzhou:Sun Yat-sen University,2008. (in Chinese)
[25] 窦克军,孙春宝.siRNA抑制A375细胞中黑色素形成[J].细胞生物学杂志,2007,29(4):617-622.
DOU K J,SUN C B.Suppression of melanin formation of A375 cells by small interfering RNA[J].Chinese Journal of Cell Biology,2007,29(4):617-622. (in Chinese)