内皮素受体在不同毛色绵羊皮肤中的表达和定位分析

Immunolocalization and Expression Analysis of Endothelin Receptor in Sheep Skin of Different Hair Color

【目的】研究内皮素受体A和B(endothelin receptor A and B,EDNR A and B)在绵羊皮肤中的表达与定位,以及在不同毛色中的差异比较,探讨其在绵羊毛色形成中的作用及其相关机制。【方法】以不同毛色的成年绵羊为研究对象(设黑色毛和白色毛两组),用RT-PCR法检测EDNR基因在不同毛色皮肤中的表达,并使用实时荧光定量PCR技术分析不同毛色绵羊皮肤EDNR基因的相对表达量,采用免疫组织化学法和Western blot法对EDNR在绵羊皮肤中的表达进行定位和定量分析。【结果】荧光定量PCR结果显示黑色绵羊中EDNRA的相对表达量是白色绵羊的1.2648倍,EDNRB的则是1.8248倍;Western blot结果显示,皮肤组织粗蛋白提取物中含有EDNR蛋白,且受体A在黑白两组皮肤中表达不显著(P>0.05),受体B在黑色组的表达显著高于白毛组(P<0.05);免疫组化实验结果显示,EDNR蛋白在两组皮肤的表皮和毛囊中均有表达,通过光密度分析,受体A在黑毛皮肤各处细胞中的表达显著高于白毛皮肤,受体B则在除毛根鞘之外的其它两处细胞中表达显著高于白毛皮肤(P<0.05)。【结论】实验结果提示,EDNR参与绵羊皮肤黑素细胞的生成,且两受体作用效应存在差异。

【Objective】 The aim of this experiment is to study the expression and immunolocalization of endothelin receptor A and B in sheep skin,to compare the expression quantity in different colors,and to explore the function of EDNR in the process of coat color formation【.Method】The mRNA and protein expression level of EDNR in sheep skin of different colors were examined by real-time quantitative PCR ,western blot and immunohistochemistry.【Result】 The EDNR A and B genes were expressed in sheep skin reliably.The expressive quantity of EDNR A gene in the black sheep skin tissue was 1.2649 times than that in white one,and the same quantity of EDNR B gene in black was 1.8248 times than that in white.By western blot there appeared EDNR proteins in total protein extracted from skin tissue,and there was no significantly difference in EDNR A between black and white skin （P0.05）,but there was higher level of EDNR B in black skin than that in white one （P0.05）.Immunohistochemistrial results showed that there was an expression of EDNR protein in black and white epidemic and hair follicle sheath cells of black and white skin tissue.Furthermore,by optical density analysis,the expression of EDNR A was higher in every cell of black skin than that in white one.There was also a higher level of EDNR B in dermal papilla cells and epidemic cells of black skin tissue than that in white ones （P 0.05）.However,there was no significant difference of EDNR B in hair follicle sheath cells of black and white skin tissue.【Conclusion】 Results showed that EDNR is involved in the process of coat color formation,and a difference in the functional efficiency of the two receptors is existed.