绿色荧光蛋白-荧光素酶标记的人源化非小细胞肺癌原位动物模型建立方法  

Establishment method of in situ animal model of humanized non-small cell lung cancer labeled by green fluorescence protein-luciferase

作　　者：续畅 刘泽洲 张灿 白玥 安志静 钟晓松 XU Chang;LIU Zezhou;ZHANG Can;BAI Yue;AN Zhijing;ZHONG Xiaosong(Clinical Trial Institutions for Drug and Medical Device,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China;Hongtai Community Health Service Center,Langfang Guangyang District Jiefang Road Office,Langfang 065000,Hebei,China;Center for Clinical Gene and Cell Engineering,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China)

机构地区：[1]首都医科大学附属北京世纪坛医院药物及医疗器械临床试验机构,北京市100038 [2]河北省廊坊市广阳区解放道办事处宏泰社区卫生服务中心,河北省廊坊市065000 [3]首都医科大学附属北京世纪坛医院临床基因与细胞工程中心,北京市100038

出　　处：《广西医学》2023年第18期2223-2227,2243,共6页Guangxi Medical Journal

基　　金：北京市科技计划课题(Z161100000216136)。

摘　　要：目的采用绿色荧光蛋白-荧光素酶(GL)标记的人类非小细胞肺癌(NSCLC)细胞建立人源化NSCLC原位动物模型。方法制备GL逆转录病毒液。将上述病毒液转染至处于对数生长期的人类NSCLC细胞株(H460细胞)后进行流式分选,从而构建稳定的GL阳性H460细胞(H460-GL细胞)。将15只Nod-Scid小鼠随机分为低剂量组(n=5)、中剂量组(n=5)、高剂量组(n=5),分别以H460-GL细胞1.0×10^(4)个/20μL、1.0×10^(6)个/20μL、1.0×10^(8)个/20μL的密度进行小鼠肺组织原位注射。实验期间观察各组小鼠的一般情况及存活情况。每周通过荧光活体成像观察各组小鼠体内肿瘤的成活、生长情况。造模后第35天处死成瘤小鼠,取肺组织进行病理组织学观察。结果(1)大部分小鼠出现呼吸系统肿瘤相关症状;濒死期小鼠胸腔内均出现不规则形状的白色肿瘤组织,且其与周围肺组织及心脏粘连严重。(2)造模后第22天开始各组小鼠逐渐死亡,于造模后第35天低剂量组、中剂量组、高剂量组小鼠的存活率分别为80%(4/5)、20%(1/5)、0。(3)在低剂量组中,于造模后第14天仅有1只小鼠出现H460-GL细胞荧光,造模第28天的成瘤率为60%(3/5),且成瘤小鼠体内的荧光强度大小不一;在中剂量组、高剂量组中,所有小鼠在造模后第7天均出现H460-GL细胞荧光,且荧光均逐渐增强,在造模后第14天、第21天成瘤小鼠体内的荧光强度相似且较为均匀。结论该造模方法可获得稳定的人源化NSCLC原位动物模型,且操作简便、易于复制,可在动物体内模拟人类NSCLC发生、发展、转移过程,并能实时观察肿瘤大小与转移路径。Objective To establish the in situ animal model of humanized non-small cell lung cancer(NSCLC)by employing human NSCLC cells labeled by green fluorescence protein-luciferase(GL).Methods GL retroviral venom was prepared.The aforementioned venom was transfected into human NSCLC cell strain(H460 cells)in logarithmic growth phase and then sorted by the fluorescence-activated cell sorting,so as to establish stable GL positive H460 cells(H460-GL cells).A total of 15 Nod-Scid mice were randomly assigned to low-dose group(n=5),medium-dose group(n=5),or high-dose group(n=5),and the in situ injection of H460-GL cells in pulmonary tissues was given to mice at the density of 1.0×10^(4)/20μL,1.0×10^(6)/20μL,and 1.0×10^(8)/20μL,respectively.The general status and survival of mice in various groups were observed during experiment.The fluorescence in vivo imaging was used to observe survival and growth of tumors in mice of various groups every week.After 35 days of modeling,tumorigenic mice were killed,and pulmonary tissues were obtained for histopathological observation.Results(1)Most of the mice developed respiratory system tumor-associated symptoms.White tumor tissues in irregular shape appeared in thoracic cavity of the dying mice,which adhered seriously to surrounding pulmonary tissues and heart.(2)On the 22 nd day after modeling,mice in various groups gradually died.The survival rates of mice in the low-,medium-,and high-dose groups were 80%(4/5),20%(1/5),and 0 on the 35 th day after modeling.(3)In the low-dose group,H460-GL cytofluorometer appeared in only 1 mouse on the 14 th day after modeling,and the tumor formation rate was 60%(3/5)on the 28 th day after modeling,and the fluorescence intensity in tumorigenic mice was not of uniform size.In the medium-and high-dose groups,H460-GL cytofluorometer appeared in all mice on the 7 th day after modeling,and fluorescence was gradually enhanced;furthermore,the fluorescence intensity of tumorigenic mice was similar and more uniform on the 14 th and 21 st day after modeling

关 键 词：非小细胞肺癌 原位动物模型 人源化 绿色荧光蛋白-荧光素酶 逆转录病毒 荧光活体成像 

分 类 号：R734.2[医药卫生—肿瘤]