YAP对梅花鹿鹿茸间充质细胞增殖与分化的影响  

Effects of YAP on proliferation and differentiation of sika deer antler mesenchymal cells

作　　者：张静 柳舒 黄吉成 岳占碰 杨占清 郭斌 ZHANG Jing;LIU Shu;HUANG Jicheng;YUE Zhanpeng;YANG Zhanqing;GUO Bin(College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医学报》2021年第2期315-321,共7页Chinese Journal of Veterinary Science

基　　金：吉林省自然科学基金资助项目(20180101247JC);国家自然科学基金资助项目(31873003)。

摘　　要：以梅花鹿为研究对象,通过流式细胞术、荧光定量PCR等方法,探讨YAP(Yes-associated protein,即Yes相关蛋白)对鹿茸间充质细胞增殖与分化的影响。分离培养鹿茸间充质细胞,添加一定浓度的YAP抑制剂Verteporfin后,通过流式细胞术检测发现,细胞的增殖活性被显著抑制,G0/G1期细胞百分比增加,S期细胞百分比降低。同时,Verteporfin可降低CCND1、CCND2、CCND3、CCNE1、CDK2、CDK4和CDK6在鹿茸间充质细胞中的表达。Verteporfin处理12,24,36,48,72 h后,软骨细胞标志性分子COLⅡ、SOX9和AGC的表达显著增强。进一步研究了YAP对间充质细胞中活性氧(reactive oxygen species,ROS)的影响,结果显示Verteporfin可增加间充质细胞中ROS含量,促使细胞中超氧阴离子(O2-)和线粒体ROS(mtROS)含量升高。为了探究YAP是否通过ROS来影响鹿茸间充质细胞的分化,细胞经Verteporfin处理2 h后再添加抗氧化剂GSH和NAC处理24 h,结果显示GSH和NAC可显著减低Verteporfin对COLⅡ、SOX9和AGC mRNA的诱导效应。结果表明,YAP在鹿茸间充质细胞的增殖与分化过程中起重要作用。In this experiment,sika deer was used as the research object to study the effects of YAP(Yes-associated protein)on the proliferation and differentiation of antler mesenchymal cells by flow cytometry assay and fluorescence quantitative PCR.After antler mesenchymal cells were isolated,cultured and treated with a certain concentration of YAP inhibitor Verteporfin.The result of flow cytometry assay showed that cell proliferation activity reduced significantly,the percentage of cells in G0/G1 phase increased,and the percentage of cells in S phase decreased.Meanwhile,Verteporfin reduced the expression of CCND1,CCND2,CCND3,CCNE1,CDK2,CDK4 and CDK6 in antler mesenchymal cells.After treatment with Verteporfin for 12,24,36,48 and 72 h,the expression of chondrocyte markers COLⅡ,SOX9 and AGC was significantly enhanced.Furthermore,the effect of YAP on ROS(reactive oxygen species)in mesenchymal cells was investigated.The result revealed that Verteporfin increased the content of ROS in mesenchymal cells,causing the elevation of superoxide anion(O2-)and mitochondrial ROS(mtROS)content.To explore whether ROS mediates the effects of YAP on antler mesenchymal cell differentiation,cells were treated with Verteporfin for 2 h and then added with antioxidants GSH and NAC for 24 h.The results showed that GSH and NAC could significantly reduce the inductive effects of verteporfin on COLⅡ,SOX9 and AGC mRNA.In conclusion,YAP plays an important role in the proliferation and differentiation of antler mesenchymal cells.

关 键 词：YAP VERTEPORFIN 鹿茸 间充质细胞 增殖 分化 

分 类 号：S852.2[农业科学—基础兽医学] S865.4[农业科学—兽医学]