永生化人巩膜成纤维细胞系建立的研究  

Establishment of immortalized human scleral fibroblast cell line

作　　者：李佳颖 李芃 程祯 王文斌 李仕明[1] 田佳鑫[1] 齐越[1] 钱俞羊 刘子涵 王宁利[1] Li Jiaying;Li Peng;Cheng Zhen;Wang Wenbin;Li Shiming;Tian Jiaxin;Qi Yue;Qian Yuyang;Liu Zihan;Wang Ningli(Beijing Institute of Ophthalmology,BeijingTongren Eye Center,Beijing Tongren Hospital,Capital Medical University,Beijing Keynote Laboratory of Ophthalmology and Visual Science,Beijing 100730,China;School of Life Sciences,Nankai University,Tianjin 300071,China)

机构地区：[1]首都医科大学附属北京同仁医院、北京同仁眼科中心、北京市眼科研究所眼科学与视觉科学北京市重点实验室,100730 [2]南开大学生命科学学院,300071

出　　处：《眼科》2023年第5期387-391,共5页Ophthalmology in China

基　　金：国家自然科学基金面上项目(82071000)。

摘　　要：目的建立永生化的人巩膜成纤维细胞系,并利用转录组测序分析永生化的影响。设计实验研究。研究对象实验组为永生化人巩膜成纤维细胞,对照组为原代人巩膜成纤维细胞。方法采用携带SV40 T基因质粒及lenti-Mix慢病毒包装质粒,利用磷酸钙转染法转染HEK293T获得慢病毒颗粒。用获得的慢病毒颗粒转染原代细胞,暴露于含有1μg/ml嘌呤霉素中2天,存活的细胞继续在培养基中体外培养至25代,获得永生化的人巩膜成纤维细胞系。之后,对永生化前后的细胞进行RNA提取及测序,分析永生化细胞与原代细胞转录本特性。使用DESeq包分析差异基因,并使用Clusterprofiler进行基因本体(GO)分析。主要指标细胞形态、胶原蛋白相关基因表达量和转录本相关性。结果永生化人巩膜成纤维细胞保持了原代细胞的形态,并呈现显著增强的增生能力,成功培养20代以上。永生化细胞与原代细胞的转录组高度相似,RNA测序显示两者相关性为r2=0.995。基于DESeq包的差异基因分析显示转录本共比对出22930个基因及33691个异构体,其中有2063(3.6%)个基因的表达呈上调趋势,2776(4.9%)个基因的表达呈下调趋势。GO功能分析结果显示,上调的基因主要聚集在DNA复制、细胞分裂等细胞基础代谢活动及与病毒的相互作用,下调的基因主要富集于细胞外基质调节与细胞间连接等通路。细胞外基质相关基因通路中,编码胶原蛋白、纤维蛋白基因转录略微下调(log2倍数变化=-0.4~-3.1),COL1A1转录量下调3.34倍。结论本研究中利用慢病毒转染法通过导入SV40T外源基因的方式构建了永生化的人巩膜成纤维细胞系,与原代细胞相似性高,稳定性好,可为巩膜相关实验提供充足的研究材料。Objective To establish an immortalized human scleral fibroblast cell line and analyze the effect of immortalization using transcriptome sequencing.Design Experimental study.Participants The experimental group was immortalized human scleral fibroblasts,and the control group was primary human scleral fibroblasts.Methods Lentiviral particles were obtained by transfection of HEK293T using calcium phosphate transfection method using plasmid carrying SV40 T gene and lenti-Mix lentivirus packaging plasmid.The obtained lentiviral particles were transfected with primary cells and exposed to puromycin containing 1μg/ml for 2 days,and the surviving cells were continued to be cultured for 25 generations to obtain immortalized human scleral fibroblast cell lines.Afterward,total RNA of primary human scleral fibroblasts and immortalized human scleral fibroblasts were extracted and sequenced to analyze the transcript characteristics of immortalized cells and primary cells.Differential genes were analyzed using the DESeq package and gene ontology(GO)analysis was performed using Clusterprofiler.Main Outcome Measures Cell morphology,collagen-related gene expression and transcript correlation.Results Immortalized human scleral fibroblasts maintained the morphology of the primary cells and showed significantly enhanced proliferation ability and were successfully cultured for more than 20 generations.The transcriptomes of the immortalized and primary cells were highly similar,and the correlation between the two was r2=0.995 obtained by RNA sequencing.The transcriptome yielded 22903 genes and 33691 isoforms,and the eifferential gene analysis based on DESeq package revealed that 2063(3.6%)genes were up-regulated and 2776(4.9%)genes were down-regulated.GO functional analysis showed that the up-regulated genes were mainly clustered in DNA replication,cell division and other basic cellular metabolic activities and interactions with viruses,while the down-regulated genes were mainly enriched in extracellular matrix regulation and intercell

关 键 词：巩膜 成纤维细胞 永生化 转录组 

分 类 号：R772.3[医药卫生—眼科]