DF3/MUC1启动子调控的重组腺病毒的构建及循环肿瘤细胞检测效果的鉴定  

Construction and evaluation of a recombinant adenovirus regulated by the DF3/MUC1 promoter for circulating tumor cell detection

作　　者：王程 顾慧慧 宣亮 郑勤 朱传东 徐瀚峰 童金龙 王礼学 WANG Cheng;GU Huihui;XUAN Liang;ZHENG Qin;ZHU Chuandong;XU Hanfeng;TONG Jinlong;WANG Lixue(Department of Radiation Center,Nanjing Hospital Affiliated to Nanjing University of Chinese Medicine(the Second Hospital of Nanjing),Nanjing 210003,China;Department of Oncology,Nanjing Hospital Affiliated to Nanjing University of Chinese Medicine(the Second Hospital of Nanjing),Nanjing 210003,China)

机构地区：[1]南京中医药大学附属南京医院(南京市第二医院)放疗中心,江苏南京210003 [2]南京中医药大学附属南京医院(南京市第二医院)肿瘤科,江苏南京210003

出　　处：《南京医科大学学报（自然科学版）》2024年第9期1190-1197,1226,共9页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省青年医学重点人才培养项目(QNRC2016054)。

摘　　要：目的:构建含DF3/MUC1启动子转录调控序列和绿色荧光蛋白(green fluorescent protein,GFP)的重组腺病毒(Ad-DF3-copGFP),探讨其在循环肿瘤细胞(circulating tumor cell,CTC)检测方面的作用。方法:制备并纯化重组腺病毒(Ad-DF3-copGFP),与本实验室构建保存的重组腺病毒(Ad-hTERT-copGFP)进行对比研究,通过感染肺腺癌A549、H1299细胞及健康人外周血单核细胞(peripheral blood mononuclear cell,PBMC)以评估感染效率与非特异性感染率;将A549细胞按一定数量加入到健康人外周血中来模拟CTC,使用两种重组病毒感染并检测模拟CTC细胞以进行检出率测定;使用Ad-DF3-copGFP法及Ad-hTERT-copGFP法检测肺癌患者临床样本中的CTC,初步评估临床CTC检测的效果。结果:成功构建了重组腺病毒Ad-DF3-copGFP,其对A549、H1299细胞有较高感染效率,且与Ad-hTERT-copGFP相比,Ad-DF3-copGFP非特异性感染率更低(P<0.001);Ad-DF3-copGFP法模拟CTC检出率(77.3%)高于Ad-hTERT-copGFP法(69.6%);在临床CTC检测中,Ad-DF3-copGFP法CTC检出数[每4 mL(10.90±2.42)个]明显高于Ad-hTERT-copGFP法[每4 m L(6.20±1.81)个,P<0.001]。结论:成功构建重组腺病毒(Ad-DF3-copGFP),其检测CTC具有可靠性和高效性,为CTC检测提供了新方法。Objective:To construct a recombinant adenovirus(Ad-DF3-copGFP)containing the DF3/MUC1 promoter transcriptional regulatory sequence and green fluorescent protein(GFP)and to investigate its role in the detection of circulating tumor cells(CTC).Methods:A recombinant adenovirus(Ad-DF3-copGFP)was prepared and purified.Comparative studies were conducted with a previously constructed and stored recombinant adenovirus(Ad-hTERT-copGFP)in our laboratory.Infection efficiency and non-specific infection rates were evaluated by infecting lung adenocarcinoma A549 and H1299 cells,as well as peripheral blood mononuclear cells(PBMC)from healthy individuals.A549 cells were artificially added to healthy peripheral blood to simulate CTC,and the infection with both recombinant viruses was used to detect simulate CTC for detection rate determination.The Ad-DF3-copGFP and Ad-hTERTcopGFP methods were used to detect CTC in clinical samples from lung cancer patients,and the initial clinical CTC detection performance was evaluated.Results:The recombinant adenovirus(Ad-DF3-copGFP)was successfully constructed,showing a high infection efficiency for both A549 and H1299 cells.Compared with Ad-hTERT-copGFP,Ad-DF3-copGFP showed a lower non-specific infection rate(P<0.001).The overall detection rate using the Ad-DF3-copGFP method(77.3%)was higher than that using the AdhTERT-copGFP method(69.6%).In clinical CTC detection,the number of CTC detected by the Ad-DF3-copGFP method[(10.90±2.42)cells per 4 mL]was significantly higher than that by the Ad-hTERT-copGFP method[(6.20±1.81)cells per 4 mL,P<0.001].Conclusion:The recombinant adenovirus(Ad-DF3-copGFP)is successfully constructed,demonstrating a reliable and efficient detection of CTC,thus providing a new method for CTC detection.

关 键 词：循环肿瘤细胞 DF3/MUC1 腺病毒 

分 类 号：R730.4[医药卫生—肿瘤]