MDCK无成瘤性悬浮细胞株的蛋白组学差异分析  

Differential Proteomic Analysis of MDCK Non-tumorigenic Suspension Cell Line

作　　者：刘耿 裴梦源 仇真毓 唐甜 何婷[1,2] 乔自林[1,2,3] 王家敏[1,2,3] 刘振斌 LIU Geng;PEI Meng-Yuan;QIU Zhen-Yu;TANG Tian;HE Ting;QIAO Zi-Lin;WANG Jia-Min;LIU Zhen-Bin(Gansu Tech Innovation Center of Animal Cell,Northwest Minzu University,Lanzhou 730030,China;Engineering Research Center for Key Technologies and Industrialization of Cell-Based Vaccines,Ministry of Education,Northwest Minzu University,Lanzhou 730030,China;Biomedical Research Center/Key Laboratory of Bioengineering and Technology State Ethnic Affairs Commission,Northwest Minzu University,Lanzhou 730030,China)

机构地区：[1]西北民族大学甘肃省动物细胞技术创新中心,兰州730030 [2]西北民族大学细胞基质疫苗关键技术与产业化教育部工程研究中心,兰州730030 [3]西北民族大学生物医学研究中心/生物工程与技术国家民委重点实验室,兰州730030

出　　处：《农业生物技术学报》2025年第3期616-627,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(32160164);中央高校基本科研业务费项目(31920230001,31920240077);甘肃省科技计划(22JR11RA239)。

摘　　要：Madin-Darby犬(Canis lupus familiaris)肾(Madin-Darby canine kidney, MDCK)细胞作为流感疫苗生产的基质细胞,具有标准化、高产量的优点,但是其贴壁生长阻碍了生产规模的扩大,其成瘤性阻碍了MDCK基质疫苗的推广。本研究通过单克隆筛选和悬浮驯化成功获得1株MDCK无成瘤性悬浮细胞株XF06。以XF06和成瘤性悬浮细胞株XF04进行裸鼠(Mus musculous)荷瘤实验,将1×107个细胞皮下注射到无胸腺裸鼠的背部,每组10只;以人(Homo sapiens)宫颈癌细胞系(Hela)(有成瘤性)作为阳性对照,以人胚肺成纤维细胞(MRC5)(无成瘤性)作为阴性对照。结果显示,筛选的细胞株XF06在裸鼠体内未形成肿瘤。利用数据非依赖性采集(data-independent acquisition, DIA)蛋白组学技术比较XF04和XF06的蛋白表达差异,筛选细胞成瘤相关基因,并进行基因和蛋白表达水平的验证。结果显示,4个蛋白(含杆状病毒IAP重复序列蛋白5 (baculoviral IAP repeat containing 5, BIRC5)、肌动蛋白结合蛋白1 (fascin actinbundling protein 1, FSCN1)、胰岛素样生长因子2 mRNA结合蛋白1 (insulin like growth factor 2 mRNA binding protein 1, IGF2BP1)、肿瘤坏死因子α诱导蛋白2 (TNF alpha induced protein 2, TNFAIP2))在无成瘤性的XF06细胞中的基因和蛋白表达水平均显著降低(P<0.05),可能参与MDCK细胞成瘤的调控。本研究为利用基因编辑技术抑制细胞成瘤的靶基因筛选提供参考依据。Madin-Darby canine(Canis lupus familiaris)kidney(Madin-Darby canine kidney,MDCK)cells have the advantages of standardization and high yield as stromal cells for influenza vaccine production.However,its adherent growth has hindered the scale-up of production and its tumorigenicity has prevented the dissemination of MDCK-based vaccines.This study successfully obtained a non tumorigenic suspension cell line XF06 of MDCK through monoclonal screening and suspension domestication.A nude mouse(Mus musculous)tumor experiment was conducted using XF06 and tumorigenic suspension cell line XF04.1×107 cells were subcutaneously injected into the back of thymus free nude mice,with 10 mice in each group;Using human(Homo sapiens)cervical cancer cell lines(Hela)(with tumorigenicity)as positive controls and human embryonic lung fibroblasts(MRC5)(without tumorigenicity)as negative controls.The results showed that the screened cell line XF06 did not form tumors in nude mice.The protein expression difference of XF04 and XF06 was compared by data-independent acquisition(DIA)proteomic technology,and the tumorigenic genes were screened,and the expression levels of genes and proteins were verified.The results shows that the gene and protein expression levels of 4 proteins(baculoviral IAP repeat containing 5(BIRC5),fascin actin-bundling protein 1(FSCN1),insulin like growth factor 2 mRNA binding protein 1(IGF2BP1),TNF alpha induced protein 2(TNFAIP2))were significantly reduced in non-tumorigenic XF06 cells(P<0.05),which might be involved in the regulation of tumorigenic MDCK cells.This study provides reference for the screening of target genes using gene editing technology to inhibit tumorigenesis.

关 键 词：Madin-Darby犬肾(MDCK)细胞 无成瘤性 悬浮细胞株 蛋白质组学 基因工程疫苗细胞系 

分 类 号：S85[农业科学—兽医学] Q813.11[农业科学—畜牧兽医]