AGRP 基因在山羊组织表达及其对黑色素生成的作用机制

Expression of AGRP Gene in Goat Tissue and Its Action Mechanism on Melanin Production

为了阐明AGRP基因在山羊皮肤表达模式以及对黑色素生成的作用机制。本研究通过qRT-PCR法检测AGRP基因在3只酉州乌羊不同组织和3只板角山羊皮肤组织表达情况。同时,通过体外试验分析不同浓度外源性AGRP蛋白对黑色素细胞影响,利用EdU法检测细胞活力、环磷腺苷(cyclic adenosine monophosphate,cAMP)试剂盒检测cAMP活性、并检测其对真黑色素含量和黑素生成相关基因黑素皮质素1型受体(melanocortin 1 receptor,MC 1R)、酪蛋白酶(tyrosinase,TYR)、酪蛋白酶相关蛋白酶1(tyrosinase related protein 1,TYRP 1)、多巴色素互变异构酶(dopachrome tautomerase,DCT)mRNA表达的影响。结果表明,AGRP基因在酉州乌羊组织均有表达,其中在垂体中表达最高,与其他组织相比差异显著(P<0.05);板角山羊与酉州乌羊皮肤中AGRP表达量差异极显著(P<0.05)。添加不同浓度AGRP蛋白对黑色素生成和黑色素细胞增殖影响不显著(P>0.05);随着AGRP浓度的升高,黑色素生成通路中cAMP活性显著下降(P<0.05);200 nmol·L^(-1) AGRP显著抑制MC1 R mRNA表达且促进TYR、TYRP1 mRNA表达(P<0.05);添加不同浓度外源性α-MSH均显著(P<0.05)挽救AGRP对黑色素细胞功能的影响。AGRP基因与酉州乌羊皮表型密切相关,确定AGRP在黑色素生成过程中的生物学功能为深入揭示AGRP对酉州乌羊皮肤黑色素沉积的作用机制奠定基础。

In order to elucidate the expression pattern of AGRP gene in goat skin and its action mechanism on melanin production.The tissue expression level of AGRP gene in different tissues of Youzhou dark goats and skin tissue of 3 Banjiao goats were detected by qRT-PCR.And the effects of exogenous AGRP protein at different concentrations on melanocytes were analyzed by in vitro experiments.The cell viability was detected by EdU method,and cAMP activity was detected by cyclic adenosine monophosphate kit,as well as its effects on the content of eumelanin and the mRNA expression of melanin-related genes MC 1R,TYR,TYRP 1 and DCT.This results showed that AGRP gene was expressed in different tissues of Youzhou dark goats and its expression in the pituitary gland was the highest(P<0.05).Meanwhile,the expression level of AGRP in the skin of Banjiao goat and Youzhou dark goat were significantly different(P<0.05).The addition of different concentrations of AGRP protein had no significant effect on the melanin production and proliferation of melanocytes(P>0.05).cAMP activity in the melanin production pathway significantly decreased(P<0.05)with the increasing of AGRP concentration.The 200 nmol·L^(-1) AGRP significantly inhibited the expression of MC1 R mRNA and enhanced the mRNA expression of TYR and TYRP1(P<0.05).The addition of exogenousα-MSH at different concentrations could significantly(P<0.05)salvage the effects of AGRP on function of melanocyte.AGRP gene is closely related to that phenotype of Youzhou dark goat skin.Determining the biological function of AGRP in melanin production can lay the foundation for further revealing the mechanism of AGRP on melanin deposition in skin of Youzhou dark goat.