转录因子ID1对绵羊卵泡颗粒细胞增殖和凋亡的影响  

Effects of Transcription Factor ID1 on Proliferation and Apoptosis of Sheep(Ovis aries)Follicular Granulosa Cells

作　　者：张配颖 宋鹏琰 周荣艳[1] 付强[1] 周营 丁虹 ZHANG Pei-Ying;SONG Peng-Yan;ZHOU Rong-Yan;FU Qiang;ZHOU Ying;DING Hong(College of Animal Science and Technology,Hebei Agricultural University,Baoding 071001,China)

机构地区：[1]河北农业大学动物科技学院,保定071001

出　　处：《农业生物技术学报》2023年第8期1641-1650,共10页Journal of Agricultural Biotechnology

基　　金：河北省自然科学基金(C2019204039);河北农业大学精准畜牧学科群(1090064)。

摘　　要：DNA结合抑制因子1(inhibitor of DNA binding 1,ID1)在机体内广泛存在,影响细胞的增殖和凋亡。为揭示转录因子ID1在绵羊(Ovis aries)卵泡颗粒细胞增殖和凋亡过程中的作用,本研究构建了真核过表达载体pcDNA3.1-ID1,设计合成了ID1 RNA干扰片段(siID1),将分离培养的绵羊卵泡颗粒细胞进行卵泡刺激素(follicle-stimulating hormone,FSH)处理,24 h后利用qRT-PCR检测ID1基因表达。结果显示,FSH处理细胞后,ID1基因表达量极显著降低(P<0.01)。进一步将pcDNA3.1、pcDNA3.1-ID1、siID1和siNC(siID1的阴性对照)转染颗粒细胞,采用CCK-8法检测转染5 d内颗粒细胞的增殖,转染48 h后,采用JC-1试剂盒检测颗粒细胞的凋亡,并利用qRT-PCR检测增殖与凋亡相关基因的表达变化。结果显示,过表达ID1的颗粒细胞存活率降低,JC-1绿色荧光增强,增殖相关基因周期蛋白依赖性激酶2(cyclin dependent kinase 2,CDK2)、周期蛋白依赖性激酶4(cyclin dependent kinase 4,CDK4)和细胞周期素D2(cyclin D2,CCND2)的表达量显著降低(P<0.05),凋亡相关基因B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl2)的表达量显著降低(P<0.05),Bcl2相关X蛋白(Bcl2-associated X protein,Bax)基因表达量显著升高(P<0.05),胱天蛋白酶3(caspase-3,Casp3)基因表达量极显著升高(P<0.01);抑制ID1表达颗粒细胞存活率升高,JC-1绿色荧光减弱,增殖相关基因CDK2和CCND2的表达量极显著升高(P<0.01),CDK4基因表达量显著升高(P<0.05),凋亡相关基因Bcl2的表达量极显著升高(P<0.01),Bax基因表达量显著降低(P<0.05)。综上所述,转录因子ID1抑制了绵羊颗粒细胞的增殖且促进了颗粒细胞的凋亡,本研究结果为探讨ID1在卵泡颗粒细胞中的生物学功能及揭示其在卵泡发育中的作用提供了基础资料。Inhibitor of DNA binding 1(ID1)is widely present in the body,and affects cell proliferation and apoptosis.To reveal the role of transcription factor ID1 in ovarian follicular granulosa cells of sheep(Ovis aries),the eukaryotic overexpression vector pcDNA3.1-ID1 was constructed and ID1 RNA interference fragment(siID1)was designed and synthesized.The expression level of ID1 gene was detected by qRT-PCR after 24 h of isolated and cultured ovarian follicular granulosa cells treated with follicle-stimulating hormone(FSH).The results showed that the expression of ID1 gene was extremely significantly decreased after FSH treatment(P<0.01).CCK-8 assay was used to detect the proliferation of granulosa cells within 5 d after transfection with pcDNA3.1,pcDNA3.1-ID1,siNC and siID1 in granulosa cells.After 48 h of transfection,JC-1 assay was used to detect the apoptosis of granulosa cells and the expression levels of proliferation and apoptosis genes were detected by qRT-PCR.The result showed that the survival rate of ID1 overexpressed granulosa cells was reduced,the green fluorescence of JC-1 was enhanced,the expression levels of proliferation-related genes cyclin dependent kinase 2(CDK2)、cyclin dependent kinase 4(CDK4)and cyclin D2(CCND2)were significantly down-regulated(P<0.05),the expression of apoptosis-related gene B-cell lymphoma-2(Bcl2)was significantly decreased(P<0.05),Bcl2-associated X protein(Bax)gene expression was significantly increased(P<0.05),and caspase-3(Casp3)gene expression was extremely significantly increased(P<0.01).The higher survival rate of granulosa cells and the lower green fluorescence of JC-1 were found in cells with inhibited ID1 expression.The expression levels of proliferation-related genes CDK2 and CCND2 were significantly increased(P<0.01),CDK4 gene was significantly increased(P<0.05),the expression of Bcl2 was significantly increased(P<0.01),and the expression of Bax was significantly decreased(P<0.05).In summary,transcription factor ID1 inhibited the proliferation and promoted the apopto

关 键 词：DNA结合抑制因子1(ID1) 绵羊 颗粒细胞 增殖 凋亡 

分 类 号：S826[农业科学—畜牧学]