细胞羧肽酶1调控小鼠睾丸初级精母细胞GC-2增殖的作用与机制研究  

Effect and mechanism of cytosolic carboxypeptidase 1 on the proliferation of mouse primary spermatocyte GC-2 cells

作　　者：张婷 王万伦 刘桐佳 刘爽[1] 边艳超[1] 张传领 肖瑞[1] ZHANG Ting;WANG Wanlun;LIU Tongjia;LIU Shuang;BIAN Yanchao;ZHANG Chuanling;XIAO Rui(Key Laboratory of Molecular Pathology of Inner Mongolia Autonomous Region,Inner Mongolia Medical University,Inner Mongolia Autonomous Region,Hohhot 010059,China;Department of Pharmacy,Inner Mongolia Medical University,Mongolia Autonomous Region,Hohhot 010110,China)

机构地区：[1]内蒙古医科大学内蒙古自治区分子病理学重点实验室,内蒙古呼和浩特010059 [2]内蒙古医科大学药学院,内蒙古呼和浩特010110

出　　处：《中国医药导报》2022年第32期10-13,30,共5页China Medical Herald

基　　金：国家自然科学基金资助项目(31760304);教育部“春晖计划”合作科研项目(教外司留〔2019〕1383号);内蒙古自治区科技计划项目(2019GG154);内蒙古自治区自然科学基金项目(2020LH03016)。

摘　　要：目的探讨细胞羧肽酶1(CCP1)在小鼠睾丸初级精母细胞(GC-2)增殖中的作用及分子机制。方法体外正常培养GC-2细胞,利用含有空载质粒与CCP1重组质粒的慢病毒感染GC-2细胞构建阴性对照组(NC组)与CCP1过表达组(GC-2+CCP1组)细胞。CCK-8法与流式细胞术分别检测CCP1过表达对GC-2细胞增殖与周期的影响;实时荧光定量PCR检测细胞周期蛋白依赖性激酶2(Cdk2)、细胞周期蛋白B1(Ccnb1)和细胞周期蛋白D1(Ccnd1)的表达;Western blot检测Cdk2蛋白表达。结果CCK-8与流式细胞术结果显示,GC-2+CCP1组细胞的体外分裂增殖能力低于NC组,并发生了G_(2)/M期阻滞,差异有统计学意义(P<0.05)。GC-2+CCP1组细胞的CDK2 mRNA与蛋白表达高于NC组、Ccnb1与Ccnd1 mRNA低于NC组,差异均有统计学意义(P<0.05)。结论CCP1过表达可能通过影响细胞周期因子Cdk2、Ccnb1与Ccnd1的表达抑制GC-2细胞体外增殖能力发生G_(2)/M期阻滞。Objective To investigate the role of cytosolic carboxypeptidase 1(CCP1)in the proliferation of mouse testicular primary spermatocytes(GC-2)and its molecular mechanism.Methods GC-2 cells of mouse were cultured in vitro,the cells of negative control group(NC group)and CCP1 overexpression group(GC-2+CCP1 group)were constructed by infecting GC-2 cells with lentivirus containing empty plasmid and CCP1 recombinant plasmid.The influence of CCP1 overexpression on the proliferation and cycle of GC-2 cells were detected by CCK-8 and flow cytometry;the changes of cycle key regulatory proteins in GC-2+CCP1 cells,including cyclin-dependent kinase 2(Cdk2),cyclin B1(Ccnb1)and cyclin D1(Ccnd1)were detected by real-time fluorescence quantitative PCR;the changes of Cdk2 in GC-2+CCP1 cells were analyzed by Western blot.Results CCK-8 and flow cytometry showed that the proliferation ability of GC-2+CCP1 cells in vitro was lower than NC group and cycle of GC-2+CCP1 cells arrest in G_(2)/M phase,the differences were statistically significant(P<0.05).The mRNA and protein of Cdk2 in GC-2+CCP1 cells was higher than NC group and Cyclin B1 and Cyclin D1 mRNA were lower,the differences were statistically significant(P<0.05).Conclusion Overexpression of CCP1 can block GC-2 cells in G_(2)/M phase,inhibit the proliferation of GC-2 cells in vitro and affect the expression of Cdk2,Cyclin B1,and Cyclin D1.

关 键 词：细胞羧肽酶1 小鼠睾丸初级精母细胞 细胞周期蛋白依赖性激酶2 细胞周期蛋白B1 细胞周期蛋白D1 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]