摘要
【目的】分析沉默信息调节因子1(SIRT1)在黔北麻羊不同月龄睾丸组织与12月龄性腺轴组织中的表达水平,以及对睾丸间质细胞发育及抗氧化能力的影响,为探明SIRT1基因调控睾丸间质细胞生长发育机制提供理论基础。【方法】以1、3、6、9和12月龄健康的黔北麻羊公羊为研究对象,采用实时荧光定量PCR检测不同月龄睾丸组织及12月龄性腺轴组织(下丘脑、垂体、睾丸和附睾)中SIRT1的表达量;将过表达载体pEGFP-N3-SIRT1和空载体pEGFP-N3转染至山羊睾丸间质细胞,运用CCK-8法检测细胞的增殖情况,划痕试验检测细胞迁移效率,利用实时荧光定量PCR检测细胞增殖、发育及抗氧化相关基因的表达量。【结果】SIRT1基因在3、6、9和12月龄睾丸组织中的表达水平均显著高于1月龄(P<0.05,下同),以12月龄的相对表达量最高;在性腺轴上,睾丸组织中SIRT1基因相对表达量显著高于下丘脑、垂体和附睾。CCK-8法结果显示,过表达SIRT1基因在12 h后极显著促进睾丸间质细胞的增殖(P<0.01);划痕试验发现,过表达SIRT1基因组在9和18 h的细胞迁移率均显著高于空载体组;实时荧光定量PCR结果显示,过表达SIRT1基因可显著促进睾丸发育相关基因[胰岛素样生长因子2基因(IGF2)],细胞增殖相关基因[增殖细胞核抗原基因(PCNA)]和抗氧化相关基因[超氧化物歧化酶基因(SOD)、谷胱甘肽过氧化物酶1基因(GPx1)和血红素加氧酶1基因(HO-1)]的表达,对抗氧化相关基因[核因子E2相关因子2基因(Nrf2)]的表达无显著影响(P>0.05)。【结论】SIRT1基因在黔北麻羊不同月龄的睾丸组织中均有表达。成功将过表达SIRT1基因表达在睾丸间质细胞,且可能通过提高黔北麻羊睾丸间质细胞中相关基因的表达,促进细胞增殖和发育,并提高其抗氧化能力,进而直接或间接影响睾丸间质细胞的生理功能。
【Objective】The expression levels of silent regulator 1(SIRT1)in testicular tissues of different months old and gonadal axis tissues of 12 months old Qianbei Ma goat were analyzed,and the effects of SIRT1 on the development and antioxidant capacity of testis Leydig cells were analyzed,which provided a theoretical basis for exploring the mechanism of SIRT1 gene regulating the growth and development of testis Leydig cells.【Method】The expression of SIRT1 in testis and gonadal axis(hypothalamus,pituitary,testis and epididymis)was detected by real-time fluorescence quantita‐tive PCR in healthy Qianbei Ma goat rams at 1,3,6,9 and 12 months old.The overexpression vector pEGFP-N3-SIRT1 and the empty vector pEGFP-N3 were transfected into goat testis Leydig cells.CCK-8 method was used to detect cell pro‐liferation.Scratch test was used to detect cell migration efficiency.Real-time fluorescence quantitative PCR was used to detect the expression of cell proliferation,cell development and antioxidant related genes.【Result】The expression level of SIRT1 gene in testicular tissue of 3,6 and 9 months old was significantly higher than that of 1 month old(P<0.05,the same below),and that of 12 months old was the highest.On the gonadal axis,the relative expression of SIRT1 gene in tes‐ticular tissue was significantly higher than that in hypothalamus,pituitary and epididymis.The results of CCK-8 method showed that overexpression of SIRT1 gene extremely significantly promoted the proliferation of testis Leydig cells after 12 h(P<0.01).Scratch test showed that the cell mobility of overexpression SIRT1 gene group was significantly higher than that of no-load group at 9 and 18 h.Real-time fluorescence quantitative PCR results showed that overexpression of SIRT1 gene could significantly promote testicular development related gene[insulin-like growth factor 2(IGF2)],as well as ex‐pression of cell proliferation-related genes[proliferating cell nuclear antigen(PCNA)]and antioxidant-related genes[su‐peroxide dismutase(SOD),glutathione peroxidase 1(GPx1)and heme oxygenase-1(HO-1)],the expression of nuclear factor E2 related factor 2(Nrf2)was not significantly effected(P>0.05).【Conclusion】SIRT1 gene is expressed in testicu‐lar tissue in different months of Qianbei Ma goat.The overexpressed SIRT1 gene is successfully expressed in testis Leydig cells,and it may promote cell proliferation and development and improve its antioxidant capacity by increasing the ex‐pression of related genes in Qianbei Ma goat testis Leydig cells,thus directly or indirectly affecting the physiological function of testis Leydig cells.
作者
陈家靖
陈祥
张远
骆金红
嵇桃桃
唐文
付开斌
CHEN Jia-jing;CHEN Xiang;ZHANG Yuan;LUO Jin-hong;JI Tao-tao;TANG Wen;FU Kai-bin(School of Animal Science,Guizhou University/Key Laboratory of Genetic Breeding and Breeding of Plateau Mountain Animals,Ministry of Education/Guizhou Key Laboratory of Animal Genetics,Breeding and Reproduction,Guiyang,Guizhou 550025,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2023年第12期3665-3673,共9页
Journal of Southern Agriculture
基金
国家自然科学基金项目(32260835)
贵州省科技支撑计划项目(黔科合支撑[2021]一般151)
贵州省高层次创新型人才项目(黔科合平台人才-GCC[2022]021-1)
贵州省牛羊发展科研项目(QNY20221101)。
作者简介
通讯作者:陈祥(1974-),https://orcid.org/0000-0001-6495-6851,博士,教授,主要从事动物繁殖与生物技术研究工作,E-mail:xchen2@gzu.edu.cn;第一作者:陈家靖(1996-),https://orcid.org/0009-0004-4881-553X,研究方向为动物遗传育种与繁殖,E-mail:chenjiajing511@163.com。
