陕北白绒山羊卵巢颗粒细胞分离及培养条件的优化  

Optimization of isolation and culture conditions of cvarian granulosa cells from Shaanbei White Cashmere goats

作　　者：刘娇容 魏宇新 李艳艳 宋晓越 刘锦旺 朱海鲸[1] 屈雷[1] LIU Jjaorong;WEI Yuxin;LI Yanyan;SONG Xiaoyue;LIU Jinwang;ZHU Hajing;QU Lei(Shaanbei White Cashmere Goat Engineering and Technology Research Center,Yulin University,Yulin 719000,China;Jia County Tongzhen Regional Agricultural and Animal Husbandry Technology Extension Station,Yulin 719200,China;College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China)

机构地区：[1]榆林学院/陕西省绒山羊工程技术研究中心,陕西榆林719000 [2]佳县通镇区域农牧技术推广站,陕西榆林719200 [3]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《黑龙江畜牧兽医》2024年第18期45-50,131,132,共8页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目“CDC25C基因及相关miRNAs对陕北白绒山羊产羔性能的调控研究”(32060734)。

摘　　要：为了解析颗粒细胞的生物学特性及其在卵巢排卵、母羊高产中的生物学作用,试验以陕北白绒山羊卵巢为研究对象,比较了切剖法和注射器抽吸法分离卵巢颗粒细胞,检测细胞活率;分别用DMEM、DMEM/F12和MEM培养基加入10%FBS、β巯基乙醇和1%双抗培养颗粒细胞(即DMEM组、DMEM/F12组和MEM组),用CCK-8法检测培养24 h、48 h的细胞活力,EdU掺入法检测细胞增殖,RT-PCR法检测增殖相关基因(PCNA、CyclinD1、CDK2)及凋亡相关基因(BAX、BCL-2、Caspase3)的表达。结果表明:注射器抽吸法分离卵巢颗粒细胞所得的细胞活率高于切剖法,切剖法可以获得更多的细胞数量;MEM组的细胞活力极显著高于DMEM/F12组和DMEM组(P<0.01),DMEM/F12组细胞活力在24 h、48 h时高于DMEM组但差异不显著(P>0.05)。MEM组与DMEM/F12组EdU染色阳性细胞数差异不显著(P>0.05),MEM组与DMEM/F12组均显著高于DMEM组(P<0.05);MEM组PCNA、CyclinD1基因的表达量高于其他两组,但差异不显著(P>0.05),CDK2基因的表达量极显著高于其他两组(P<0.01),DMEM/F12组和DMEM组的PCNA、CyclinD1和CDK2基因的表达量差异不显著(P>0.05)。MEM组Caspase3的表达量低于其他两组但差异不显著(P>0.05),BCL-2/BAX的相对表达量极显著高于DMEM/F12组和DMEM组(P<0.01)。说明采用抽吸法分离卵巢颗粒细胞并辅以MEM培养基培养,可以实现对陕北白绒山羊卵巢颗粒细胞的有效体外培养。In order to analyze the biological characteristics of ovarian granulosa cells and their biological role in ovarian ovulation and high yield of ewes,the ovaries in Shaanbei White Cashmere goats were selected as the study subjects,and the ovarian granulosa cells were separated by incision method and syringe aspiration method,and the cell viability was measured.Granule cells were cultured in DMEM,DMEM/F12 and MEM medium with 10%FBS,β-mercaptoethanol and 1%double antibiotic(DMEM group,DMEM/F12 group and MEM group),respectively.Cell viability was measured by CCK-8;cell proliferation was measured by EdU doping method,and the expression of proliferation-related genes(PCNA、CyclinD1、CDK2)and apoptosis related gens(BAX、BCL-2、Caspase3)were measured by RT-PCR.The results showed that the viability of ovarian granulosa cells isolated by syringe aspiration was higher than that of dissection,and the cell viability of MEM group was significantly higher than that of DMEM/F12 group and DMEM group(P<0.01).The cell viability of DMEM/F12 group was higher than that of DMEM group at 24 h and 48 h,but the difference was not significant(P>0.05).There was no significant difference in the number of EdU positive cells between MEM group and DMEM/F12 group(P>0.05),but those in MEM group and DMEM/F12 group were significantly higher than those in DMEM group(P<0.05).The expression of PCNA and CyclinD1 gene in MEM group was higher than that in the other two groups,but the difference was not significant(P>0.05).The expression of CDK2 gene was significantly higher than that in the other two groups(P<0.01).There was no significant difference in the expression of PCNA,CyclinD1 and CDK2 gene between DMEM/F12 group and DMEM group(P>0.05).The expression of Caspase3 in MEM group was lower than that in the other two groups,but the difference was not significant(P>0.05).The relative expression of BCL-2/BAX in DMEM/F12 group was significantly higher than that in DMEM/F12 group and DMEM group(P<0.01).It was suggested that the ovarian granulosa cells

关 键 词：陕北白绒山羊 卵巢 颗粒细胞 分离方法 培养液 细胞增殖 

分 类 号：S827[农业科学—畜牧学]