CAJ | 학술논문

【目的】对不同毛色绵羊皮肤中的阿黑皮素原（proopiomelanocortin，POMC）进行定位和表达差异分析，确定POMC与毛色形成的关系，进一步丰富有关哺乳动物毛色形成机制的理论知识。【方法】选取年龄相近的雄性黑色和白色成年绵羊各3只，用取皮器分别在臀部采集皮肤组织3块，对其中一块制作石蜡切片后采用免疫组织化学法确定POMC在不同绵羊皮肤中的定位，另两块置于液氮中冻存后分别提取皮肤总RNA和总蛋白，总RNA经反转录成cDNA后，经NCBI分析比对后设计POMC特异性PCR引物，采用QRT-PCR方法检测POMC基因在mRNA水平的表达差异；总蛋白经SDS-PAGE凝胶电泳后，采用Western blotting方法加入POMC多克隆抗体检测POMC在蛋白水平的表达差异，得出的数据用SPSS19.0软件进行差异显著性分析，探讨POMC对毛色形成机制的调控作用。【结果】免疫组织化学结果显示POMC在黑色和白色绵羊皮肤中均有表达，且表达部位都位于毛囊根鞘周围和表皮的皮肤角质形成层细胞内；QRT-PCR结果显示POMC在黑色绵羊皮肤中mRNA的相对表达量为2.5004±0.2577**，而在白色绵羊皮肤中POMC的mRNA的相对表达量为1.0032±0.0350，黑色绵羊皮肤中POMC的mRNA的相对表达量为白色绵羊皮肤的2.5倍，二者表达差异极显著（P＜0.01）；Western boltting结果显示黑色绵羊皮肤POMC蛋白水平相对表达量为1.5253±0.0426*，而白色皮肤中POMC蛋白水平相对表达量为1.0005±0.0180，黑色皮肤POMC蛋白表达量是白色皮肤的1.55倍（P＜0.05），两者差异显著。【结论】试验表明POMC可在黑色和白色绵羊皮肤中正常表达，且其表达位置一致，但POMC在不同毛色绵羊皮肤组织中mRNA和蛋白的表达水平存在一定的差异，表明POMC的表达量变化会在一定程度上影响毛色的生成。
【목적】대불동모색면양피부중적아흑피소원（proopiomelanocortin，POMC）진행정위화표체차이분석，학정POMC여모색형성적관계，진일보봉부유관포유동물모색형성궤제적이론지식。【방법】선취년령상근적웅성흑색화백색성년면양각3지，용취피기분별재둔부채집피부조직3괴，대기중일괴제작석사절편후채용면역조직화학법학정POMC재불동면양피부중적정위，령량괴치우액담중동존후분별제취피부총RNA화총단백，총RNA경반전록성cDNA후，경NCBI분석비대후설계POMC특이성PCR인물，채용QRT-PCR방법검측POMC기인재mRNA수평적표체차이；총단백경SDS-PAGE응효전영후，채용Western blotting방법가입POMC다극륭항체검측POMC재단백수평적표체차이，득출적수거용SPSS19.0연건진행차이현저성분석，탐토POMC대모색형성궤제적조공작용。【결과】면역조직화학결과현시POMC재흑색화백색면양피부중균유표체，차표체부위도위우모낭근초주위화표피적피부각질형성층세포내；QRT-PCR결과현시POMC재흑색면양피부중mRNA적상대표체량위2.5004±0.2577**，이재백색면양피부중POMC적mRNA적상대표체량위1.0032±0.0350，흑색면양피부중POMC적mRNA적상대표체량위백색면양피부적2.5배，이자표체차이겁현저（P＜0.01）；Western boltting결과현시흑색면양피부POMC단백수평상대표체량위1.5253±0.0426*，이백색피부중POMC단백수평상대표체량위1.0005±0.0180，흑색피부POMC단백표체량시백색피부적1.55배（P＜0.05），량자차이현저。【결론】시험표명POMC가재흑색화백색면양피부중정상표체，차기표체위치일치，단POMC재불동모색면양피부조직중mRNA화단백적표체수평존재일정적차이，표명POMC적표체량변화회재일정정도상영향모색적생성。
Objective]The objective of this study is to identify the regulation effect of POMC on coat pigment formation via analyzing the differences of POMC expression and location between different coat colors of sheep skin, in order to further enrich the theoretical knowledge about the formation mechanism of animal coat color.[Method] Three black adult sheep and three white sheep with matched age were selected, then three pieces of skin tissue from the buttocks of sheep were collected, respectively. One of them was used for immunohistochemistry assay to investigate the expression location of POMC between different coat color sheep after tissue embedded, the others were hold in liquid nitrogen for the extraction of total RNA and protein. The POMC-specific primers were designed by blasting in the NCBI database. After the reverse transcription of total RNA, QRT-PCR assay was proceeded to detect POMC mRNA expression of different coat color sheep. Then the protein expression level of POMC between different color sheep was evaluated by using Western blotting. All data were analyzed by SPSS19.0 to explore the mediate effect of POMC on coat color formation mechanism.[Result]Immunohistochemistry showed that POMC was expressed in both black and white sheep skin, and the expression sites were located around the keratinocytes of hair follicle root sheath and epidermal. QRT-PCR showed that the relative expression of POMC mRNA in black sheep skin was 2.5004±0.2577**, while in white was 1.0032±0.0350, the quantity expression of POMC mRNA level of black sheep was 2.50 times as that of white sheep (P<0.01). Western blotting result showed that the expression of POMC protein in black and white sheep were 1.5253±0.04026* and 1.0005±0.0180, respectively, the expression of POMC protein in black sheep was 1.55 times as that in white sheep (P<0.05).[Conclusion]POMC expressed normally in black and white sheep skin, and its expression was located in consistent position, the expression of POMC in black and white sheep skin have differences, which may involve in formation of coat color.