Foxj2基因对羊驼黑素细胞黑色素生成的影响  

Effects of Foxj2 on Melanogenesis in Alpaca Melanocytes

作　　者：段志成 董常生[1] 范瑞文[1] 谢建山[2] 杨玉静 聂瑞强 DUAN Zhicheng;DONG Changsheng;FAN Ruiwen;XIE Jianshan;YANG Yujing;NIE Ruiqiang(College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China;School of Basic Medicine,Shanxi Medical University,Taiyuan 030001,China)

机构地区：[1]山西农业大学动物医学学院,山西太谷030801 [2]山西医科大学基础医学院,山西太原030001

出　　处：《山西农业科学》2023年第6期702-708,共7页Journal of Shanxi Agricultural Sciences

基　　金：国家高科技研究发展计划(863计划)(2013AA102506);公益性行业(农业)科研专项(201303119);山西农业大学创新团队建设计划项目(CXTD201201)。

摘　　要：叉头框转录蛋白家族成员Foxj2(Forkhead Box J2)基因在U87细胞和U251细胞中与上皮-钙黏蛋白(E-cadherin,CDH1)的表达呈正相关,且CDH1参与黑色素的生成具有介导表皮-黑色素单位完整性的作用。为了验证Foxj2基因参与调节羊驼黑素细胞中黑色素的生成,采用PCR方法验证Foxj2基因在羊驼皮肤内的表达情况;采用生物信息学方法分析Foxj2与黑色素生成相关基因TYR以及Foxj2与黑色素的生成相关蛋白的关联性;合成Foxj2的siRNA和空载siRNA,构建沉默siRNA的羊驼黑素细胞模型,观察记录细胞的转染情况;使用Real-time PCR方法检测各组羊驼黑素细胞中Foxj2基因和黑色素生成相关基因TYR的mRNA变化;通过Western blot的方法检测比较各组细胞中Foxj2与TYR的蛋白水平的变化;提纯各组细胞的3种黑色素(总黑素、真黑素、褐黑素),使用酶标仪测各组细胞中3种黑色素的OD值并比较变化。结果表明,羊驼黑素细胞中检测到Foxj2基因表达;生物信息学分析得出,Foxj2基因在细胞内涉及运输氨基酸调控、生长因子等一系列作用,在Foxj2中存在的Forkhead结构域可以与TYR基因启动子存在结合位点;Real-time PCR结果显示,和NC组相比,沉默组中Foxj2的mRNA表达量极显著降低24990%,TYR的表达量显著升高35%;Western blot结果显示,沉默组Foxj2的蛋白相对表达水平极显著降低25%,TYR的蛋白相对表达水平极显著升高25%。酶标仪波长分析显示,与NC组相比,沉默组的总黑素相对表达水平极显著升高88.0%,真黑素相对表达水平极显著升高21.8%,褐黑素相对表达水平极显著升高21.6%。抑制羊驼黑素细胞中Foxj2基因的转录可以升高羊驼黑素细胞中黑色素的生成,并且对黑色素生成的主要调控基因TYR起促进作用,说明Foxj2基因可能在羊驼黑素细胞中对于黑色素的生成起着调控作用。Foxj2(Forkhead Box J2)gene,a member of the forkhead box transcription protein family,is positively correlated with the expression of epithelial-cadherin(E-cadherin,CDH1)in U87 cells and U251 cells,and CDH1 involved melanogenesis has a role in mediating the integrity of the epidermal-melanin unit.In order to verify that Foxj2 gene is involved in regulating melanogenesis in alpaca melanocytes,in this study,PCR method was used to verify the expression of Foxj2 gene in alpaca skin,the correlation between Foxj2 and melanogenesis related gene TYR,and Foxj2 and melanogenesis related proteins was analyzed by bioinformatics.Foxj2 siRNA and empty siRNA were synthesized,alpaca melanocyte model of silencing siRNA was constructed,and the transfection of cells was observed and recorded.Real-Time-PCR was used to detect the mRNA changes of Foxj2 gene and melanogenesis related gene TYR in the alpaca melanocytes of each group.Western blot was used to detect and compare the changes of Foxj2 and TYR protein levels in each group cell.Three kinds of melanin(total melanin,eumelanin,brown melanin)in the cells of each group were purified,and a microplate reader was used to measure the OD value of the three kinds of melanin in the cells of each group and compare changes.The results showed that Foxj2 gene expression was detected in alpaca melanocytes.Bioinformatics analysis showed that Foxj2 gene was involved in a series of functions such as amino acid transport regulation,growth factor and so on.The Forkhead domain existing in Foxj2 could have binding sites with the TYR gene promoter.Real-Time PCR results showed that compared with the NC group,the mRNA expression of Foxj2 in the silence group was extremely significantly decreased by 24990%,and the expression of TYR was significantly increased by 35%.Western-blot results showed that the protein relative expression level of Foxj2 gene in the silence group extremely significantly decreased by 25%,and the protein relative expression level of TYR extremely significantly increased by 25%.The wa

关 键 词：Foxj2 SIRNA TYR 生物信息学 羊驼黑素细胞 黑色素 

分 类 号：S829.9[农业科学—畜牧学]