摘要
随着基因编辑技术迅速发展,研究精原干细胞(spermatogonial stem cells,SSCs)对精子发生及其调控机制研究、转基因动物研发、基因治疗和不孕不育症的治疗以及珍稀物种的保护均具有重要意义。溶酶体相关细胞器生物合成复合体1亚基1(biogenesis of lysosome-related organelles complex 1subunit 1,BLOC1S1)具有抗布鲁氏菌的潜能,研究BLOC1S1对山羊SSCs的影响不仅能探究BLOC1S1促进SSCs增殖的能力,还能为其抗病育种研究提供细胞学基础。本研究通过同源重组构建BLOC1S1过表达载体,通过慢病毒包装、转染与嘌呤霉素筛选成功构建了山羊精原干细胞BLOC1S1过表达细胞株。通过实时荧光定量PCR(real time quantitative PCR,RT-qPCR)检测BLOC1S1的过表达效率为18倍,同时细胞生长曲线、流式细胞术检测细胞周期、5-乙炔基-2’脱氧尿嘧啶核苷(5-ethynyl-2’-deoxyuridine,EdU)染色等实验结果表明,BLOC1S1能显著增加山羊SSCs的增殖活性。RT-qPCR、免疫荧光染色、Western blotting结果显示与增殖相关的基因(PCNA、CDK2、CCND1)上调,同时调控精原干细胞增殖的关键基因EIF2S3Y的表达量也上调。本研究成功构建了山羊精原干细胞BLOC1S1过表达细胞株,提高其增殖能力,并且这种促增殖能力可能是通过EIF2S3Y/ERK通路实现的。本研究为探究BLOC1S1对山羊精原干细胞的调控作用奠定了细胞学基础,并为进一步研究BLOC1S1的生物学功能提供细胞平台,为繁育BLOC1S1修饰抗病山羊奠定了基础。
With the rapid development of gene editing technology,the study of spermatogonial stem cells(SSCs)holds great significance in understanding spermatogenesis and its regulatory mechanism,developing transgenic animals,gene therapy,infertility treatment and protecting rare species.Biogenesis of lysosome-related organelles complex 1 subunit 1(BLOC1S1)is believed to have anti-brucella potential.Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation,but also provides a cytological basis for disease-resistant breeding research.In this study,a BLOC1S1 overexpression vector was constructed by homologous recombination.The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging,transfection and puromycin screening.The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR(RT-qPCR).Furthermore,the results from cell growth curve analysis,flow cytometry for cell cycle detection,and 5-ethynyl-2′-deoxyuridine(EdU)staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs.The results of RT-qPCR,immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes(PCNA,CDK2,CCND1),and EIF2S3Y,a key gene regulating the proliferation of spermatogonial stem cells.These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway.In summary,this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line,which exhibited improved proliferation ability.This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1.These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.
作者
万仕成
张梦菲
陈文博
韩苗
杨栋慧
王聪亮
吴文萍
王瑜琪
李娜
朱海鲸
Ahmed Hamed Arisha
华进联
WAN Shicheng;ZHANG Mengfei;CHEN Wenbo;HAN Miao;YANG Donghui;WANG Congliang;WU Wenping;WANG Yuqi;LI Na;ZHU Haijing;Ahmed Hamed Arisha;HUA Jinlian(Shaanxi Stem Cell Engineering and Technology Research Center,College of Veterinary Medicine,Northwest A&F University,Yangling 712100,Shaanxi,China;Shaanxi Province Engineering&Technology Research Center of Cashmere Goats,Yulin University,Yulin 719000,Shaanxi,China;Faculty School of Veterinary Medicine,Badr University in Cairo,Badr 11829,Cairo,Egypt)
出处
《生物工程学报》
CAS
CSCD
北大核心
2023年第12期4901-4914,共14页
Chinese Journal of Biotechnology
基金
国家自然科学基金(32072806)
国家重点研发计划(2022YFD1302201)
陕西省畜牧科技示范和畜牧专项项目(20221086,20230978)。
