粒细胞集落刺激因子在羊成纤维细胞中的表达及对细胞增殖和凋亡的影响  

Transient Expression and the Effect on Proliferation and Apoptosis of Granule Cell Stimulating Factor in Ovarian Fibroblasts

作　　者：李闰婷 陈龙欣[2] 张丽萌[1,2] 何海迎 王泳[1] 杨若晨 段春辉[1] 刘月琴 王玉琴[3] 张英杰[1] LI RunTing;CHEN LongXin;ZHANG LiMeng;HE HaiYing;WANG Yong;YANG RuoChen;DUAN ChunHui;LIU YueQin;WANG YuQin;ZHANG YingJie(College of Animal Science and Technology,Hebei Agricultural University,Baoding 071001,Hebei;Molecular Biology Laboratory,Zhengzhou Normal University,Zhengzhou 450044;College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471023,Henan)

机构地区：[1]河北农业大学动物科技学院,河北保定071001 [2]郑州师范学院分子生物学实验室,郑州450044 [3]河南科技大学动物科技学院,河南洛阳471023

出　　处：《中国农业科学》2021年第11期2434-2444,共11页Scientia Agricultura Sinica

基　　金：国家现代农业(肉羊)产业技术体系建设专项(CARS-38和CARS-39);国家重点研发计划项目(2018YFD0502100)。

摘　　要：【目的】研究粒细胞集落刺激因子(granule cell stimulating factor,GCSF)在羊成纤维细胞体外培养中对其增殖、周期和凋亡的影响,为今后基于羊GCSF为靶标诱导全能干细胞进行分子遗传育种研究提供理论依据。【方法】将羊GCSF真核表达质粒pRTL1-GCSF和对照载体质粒pRTL1分别转染到1×10^(5)个细胞/mL的羊成纤维细胞中,培养48 h后,利用Trizol法分别提取总RNA并反转录为cDNA,通过实时荧光定量PCR检测羊GCSF在羊成纤维细胞中的瞬时表达水平。通过GCSF依赖型细胞系NFS-60,利用细胞活力检测试剂alamarBlue测定转染48 h后羊成纤维细胞培养上清中分泌表达的GCSF的生物学活性。通过HEK 293F悬浮培养细胞系真核表达分泌型羊GCSF蛋白后,用Ni-NTA凝胶对细胞表达至细胞培养基中的GCSF蛋白进行纯化,并SDS-PAGE检测。加入纯化的30 ng·mL^(-1)GCSF蛋白后在24和48 h时,通过alamarBlue测定羊成纤维细胞的增殖状态,利用流式细胞术检测羊成纤维细胞的细胞周期和凋亡变化。【结果】羊GCSF真核表达质粒转染羊成纤维细胞48 h,检测发现在羊成纤维细胞中GCSF表达量得到显著提高。在羊成纤维细胞中,转染了pRTL1-GCSF质粒的羊GCSF表达量是转染了pRTL1空载对照组的(50615.92±4738.83)倍(P<0.01);羊成纤维细胞瞬时分泌表达的含有GCSF蛋白的培养基上清加入到GCSF依赖型细胞系NFS-60后,试验组和阳性对照组中的NFS-60的荧光强度与阴性对照组和空白对照组相比显著升高(P<0.01),试验组中的NFS-60的荧光强度与阳性对照组相比均差异不显著(P>0.05),结果显示羊GCSF能显著刺激NFS-60细胞的增殖,表明在羊成纤维细胞中表达的羊GCSF具有生物学活性。用HEK 293F悬浮培养细胞系真核表达分泌型羊GCSF蛋白后,纯化得到羊GCSF蛋白。在羊成纤维细胞中添加30 ng·mL^(-1)的羊GCSF后,体外培养24和48 h,GCSF试验组与培养基稀释液对照组相比,细胞活力变化差�【Objective】The purpose of this paper is to study the transient expression of granule cell stimulating factor(GCSF)in ovarian fibroblast cells,and the influence of GCSF on proliferation,cell cycle,and apoptosis,to provide theoretical basis for molecular genetic breeding of sheep pluripotent stem cells induced by GCSF in the future.【Method】The sheep GCSF eukaryotic expression plasmid pRTL1-GCSF and the control vector plasmid pRTL1 were transfected into 1×105 cells·mL^(-1)sheep fibroblasts respectively.After 48 h of culture,the total RNA was extracted by Trizol method and reverse transcribed into cDNA.The transient expression level of sheep GCSF in fibroblasts was detected by real-time quantitative PCR.GCSF dependent cell line NFS-60 was used for the biological activity of GCSF secreted and expressed in the supernatant of sheep fibroblasts 48 hours after transfection,which was determined by cell viability detection reagent alamarBlue.The HEK 293F suspension culture was used to express the secreted GCSF protein.The GCSF protein expressed in the cell culture medium was purified by Ni-NTA resin and detected by SDS-PAGE.After adding the 30 ng·mL^(-1)purified GCSF protein,the proliferation of sheep fibroblasts was detected by alamarBlue at 24 h and 48 h,and the cell cycle and apoptosis of sheep fibroblasts were detected by flow cytometry.【Result】The expression level of GCSF in sheep fibroblasts was significantly increased after transfection for 48 h.In sheep fibroblasts,the expression level of GCSF transfected with pRTL1-GCSF plasmid was 50615.92±4738.83 of that of pRTL1 empty control group.The fluorescence intensity of NFS-60 in the experimental group and positive control group was significantly higher than that in the negative control group and blank control group(P<0.01),but there was no significant difference between the experimental group and the positive control group(P>0.05).The results showed that sheep GCSF could significantly stimulate the proliferation of NFS-60 cells,indicating that the GCSF e

关 键 词：GCSF 羊 转染 生物学活性 细胞周期 细胞凋亡 

分 类 号：S826[农业科学—畜牧学]