卵巢癌腹腔转移工具细胞的构建及其应用  

Construction of Artificial Cells with Application to Investigate Peritoneal Metastasis in Ovarian Cancer

作　　者：万里舟 杜汝沛 陈康梅 WAN Li-zhou;DU Ru-pei;CHEN Kang-mei(Medical Research Center,Sun Yat-sen Memorial Hospital,Sun Yat-sen University,Guangzhou 510120,China;School of Biomedical Sciences and Engineering,South China University of Technology,Guangzhou 511442,China;Department of Clinical Laboratory,Sun Yat-sen Memorial Hospital,Sun Yat-sen University,Guangzhou 510120,China)

机构地区：[1]中山大学孙逸仙纪念医院基础与转化医学研究中心,广东广州510120 [2]华南理工大学生物医学科学与工程学院,广东广州511442 [3]中山大学孙逸仙纪念医院检验科,广东广州510120

出　　处：《中山大学学报（医学科学版）》2023年第6期965-973,共9页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(82002698);广东省基础与应用基础研究基金(2019A1515110502)。

摘　　要：【目的】构建稳定共表达荧光素酶(Luc)和增强型绿色荧光蛋白(EGFP)的人源卵巢癌细胞SKOV3(SK-Luc-EGFP)并探究其在卵巢癌腹腔转移的体内外研究中的应用。【方法】利用重叠PCR扩增得到的Luc-T2A-EGFP基因片段,构建重组质粒pCDH-Luc-T2A-EGFP-Puro;采用三质粒慢病毒包装系统转染HEK 293T细胞,收集病毒液感染SKOV3细胞;通过嘌呤霉素筛选,流式细胞术检测,筛选高效表达EGFP的细胞(SK-Luc-EGFP),并通过体外生物发光实验验证Luc的表达。对SK-Luc-EGFP细胞进行如下应用的探究:利用流式细胞术和激光共聚焦区分SK-Luc-EGFP细胞与腹水微环境中的非肿瘤细胞,利用荧光显微镜观察SK-Luc-EGFP与间皮细胞的黏附,利用小动物活体成像仪观察SK-Luc-EGFP细胞的大网膜黏附以及体内成瘤的过程。【结果】成功构建慢病毒载体pCDH-Luc-T2A-EGFP-Puro;感染并筛选得到SK-Luc-EGFP单克隆细胞株。通过荧光显微镜和流式细胞术均能检测到EGFP的表达,细胞纯度达100%;通过流式细胞术和激光共聚焦成像可直观辨别SK-Luc-EGFP细胞与腹水微环境细胞。体外生物发光试验成功验证Luc的表达,且细胞数和生物发光信号的线性相关系数为0.9979。细胞黏附试验中通过荧光成像直接观察到SK-Luc-EGFP细胞对间皮细胞的黏附;经腹腔注射SK-Luc-EGFP细胞观察到细胞在裸鼠大网膜上的黏附;在SK-Luc-EGFP细胞建立的卵巢癌腹腔转移模型中,利用小动物活体成像仪实现对腹腔肿瘤的活体监测。【结论】SK-Luc-EGFP细胞株在卵巢癌腹腔转移的体内外研究中具有多重应用潜力。【Objective】To construct a human ovarian cancer cell line SKOV3(SK-Luc-EGFP)stably co-expressing luciferase(Luc)and enhanced green fluorescent protein(EGFP)and to explore its application in ovarian cancer research both in vitro and in vivo.【Methods】The recombinant plasmid pCDH-Luc-T2A-EGFP-Puro was constructed by introduc‐ing a Luc-T2A-EGFP fusion gene fragment amplified by Overlap PCR into plasmid vector.The three-plasmid lentivirus packaging system was transfected into HEK 293T cells and the viral supernatant was harvested to infect SKOV3 cells.SK-Luc-EGFP cell line with the highest fluorescence intensity of EGFP was obtained by puromycin selection and flow cytome‐try assessment,and the Luc expression of the cell line was subsequently validated by in vitro bioluminescent assay.SK-Luc-EGFP cells were further explored for the following applications:distinguishing SK-Luc-EGFP cells from non-tumor cells in ascites by flow cytometry and confocal microscopy;visualizing adhesion of SK-Luc-EGFP cells to mesothelial cells or omentum by fluorescence microscopy;monitoring process of SK-Luc-EGFP tumorigenesis by in vivo biolumines‐cence imaging.【Results】A recombinant lentiviral expression plasmid pCDH-Luc-T2A-EGFP-Puro was constructed and packaged into lentiviral particles that were then transfected into SKOV3 cells to generate SK-Luc-EGFP cell line.The pu‐rity of SK-Luc-EGFP cells based on EGFP expression was 100%as validated by fluorescence microscopy and flow cytome‐try;SK-Luc-EGFP cells could be visually distinguished from non-tumor cells in ascitic fluid by flow cytometry and confo‐cal imaging.Moreover,Luc expression in SK-Luc-EGFP cells was verified by in vitro bioluminescence assay,and a linear relationship with a correlation coefficient of 0.9979 was found between cell number and the bioluminescent signal.Adhe‐sion of SK-Luc-EGFP cells to mesothelial cells was directly observed by fluorescence imaging in in vitro adhesion assay;peritoneal adhesion of SK-Luc-EGFP cells to omentum was also ob

关 键 词：卵巢癌 腹腔转移 荧光素酶 增强型绿色荧光蛋白 构建 

分 类 号：R737.31[医药卫生—肿瘤]