人原代瘢痕疙瘩成纤维细胞系的建立及鉴定  

Establishment and identification of a human keloid fibroblasts cell line

作　　者：徐梦丽 王其飞 王静宜 陆语豪 秦泽莲[1] Xu Mengli;Wang Qifei;Wang Jingyi;Lu Yuhao;Qin Zelian(Department of Plastic Surgery,Peking University Third Hospital,Beijing 100191,China)

机构地区：[1]北京大学第三医院成形外科,北京100191

出　　处：《中华整形外科杂志》2024年第5期545-554,共10页Chinese Journal of Plastic Surgery

基　　金：国家自然科学基金(82172216)。

摘　　要：目的尝试构建永生化的人瘢痕疙瘩成纤维细胞(KFbs)系,并对其功能进行鉴定。方法标本取自1例2019年11月在北京大学第三医院行耳垂瘢痕疙瘩手术切除的32岁女性患者。将获得的瘢痕疙瘩去除皮下脂肪和表皮,应用组织贴壁法进行分离和培养,获得原代KFbs,以胰蛋白酶消化法进行传代培养;用SV40慢病毒感染原代KFbs,经过嘌呤霉素筛选纯化、传代扩增,获得永生化的人KFb系。对原代KFbs及其细胞系进行染色体核型分析、短串联重复序列(STR)和性别基因检测,来鉴定细胞系身份;采用CCK-8法检测原代KFbs和细胞系增殖能力,并分别用实时定量PCR(qRT-PCR)和蛋白质印迹法检测其特定基因(PGK1、ENO1、LDHA、GLUT1、TGF-β1、COL1、COL3、FN)的mRNA和蛋白表达水平。原代KFbs与细胞系间相关数据比较采用t检验,P<0.05表示差异有统计学意义。结果原代KFbs和构建的细胞系的细胞形态均呈典型的长梭形,对细胞系连续培养传代至20代,细胞形态与原代KFbs基本相似。性别基因检测显示两者均为女性,且两者的染色体核型分型良好,仍保留正常细胞的基本特征,而未发生恶性转变。STR鉴定结果显示,细胞系中未发现多等位基因,表明其为正常细胞的基因型,且该细胞系与已知细胞数据库信息均无重合。培养后24、48、72 h细胞系的增殖能力比原代KFbs分别增加了76.1%、125.8%、60.3%,其增殖速度明显较原代KFbs增快(P<0.05);细胞系中上述特定基因的mRNA及蛋白表达水平与原代KFbs相比无显著改变(P均>0.05)。结论成功建立了永生化的人KFbs系,其形态、表征和功能未发生明显改变,且其增殖速度较原代细胞增快。Objective To establish an immortalized human keloid fibroblasts(KFbs)cell line and identify its characteristics and functions.Methods The specimen was obtained from a 32-year-old female patient who underwent surgical resection of an earlobe keloid at Peking University Third Hospital in November 2019.The keloid tissue obtained was removed from the subcutaneous fat and epidermis.It was then separated and cultured using the tissue sticking method to obtain primary KFbs,which were passaged using the trypsin digestion method.After the primary KFbs were infected with an SV40 lentivirus,purified by puromycin,and passaged,a human KFbs cell line was established.Chromosomal karyotype analysis,short tandem repeat(STR)profiling,and gender gene detection were conducted to identify the primary KFbs and the cell line.The CCK-8 method was used to assess the proliferation ability of the cells.Quantitative real-time PCR(qRT-PCR)and Western blotting were used to detect the mRNA and protein expression levels of specific genes(PGK1,ENO1,LDHA,GLUT1,TGF-β1,COL1,COL3,FN).The comparative analysis of relevant data between primary KFbs and the cell line was conducted using t-test,and P<0.05 indicated statistical significance.Results The morphology of both the primary KFbs and the cell line was typically spindle-shaped.The cell line morphology was basically similar to that of the primary KFbs,which were continuously cultured and passaged for 20 generations.The gender gene(Amelogenin)detection showed both were females.The chromosome karyotyping of the primary KFbs and cell line was satisfactory,maintaining the fundamental characteristics of normal cells without undergoing malignant transformation.The STR identification results showed that no multiple alleles were found in the cell line,indicating a normal cell genotype.Furthermore,the cell line did not match any entries in known cell databases.After 24,48,and 72 hours of culture,the proliferation ability of the cell line increased by 76.1%,125.8%,and 60.3%compared to primary KFbs.The prolif

关 键 词：瘢痕疙瘩 成纤维细胞 永生化 细胞系 染色体核型鉴定 基因 

分 类 号：R622[医药卫生—整形外科]