摘要
目的检测巨噬细胞靶向表达PR39的重组腺病毒Ad-SP/PR39在抗胞内菌方面的作用。方法重组腺病毒Ad-SP/PR39经CsCl密度梯度离心纯化,用荧光显微镜观察绿色荧光蛋白GPF的表达并检测重组腺病毒的滴度。Ad-SP/PR39感染小鼠巨噬细胞RAW264.7、人结肠癌细胞株Lovo、人肝癌细胞株HepG2、人乳腺癌细胞株ZR-75-30和非洲绿猴肾细胞株COS-7后,经RT-PCR和免疫细胞化学检测PR39的靶向表达特异性。将含Ad-SP/PR39的RAW264.7细胞裂解液作用于减毒结核分枝杆菌(BCG),检测其体外杀灭结核杆菌BCG的能力。另取RAW264.7细胞,吞噬BCG后,加入Ad-SP/PR39感染该细胞并检测细胞内活菌量,以观察其细胞内杀菌作用。结果经纯化后的重组腺病毒滴度可达5×1011efu/ml,能够有效感染小鼠巨噬细胞RAW264.7等多种细胞株。RT-PCR及免疫细胞化学技术检测各株细胞转录水平表达情况显示小鼠巨噬细胞RAW264.7能高效表达抗菌肽PR39,而其他细胞株几乎没有表达。与感染对照腺病毒Ad-C的RAW264.7细胞相比,转染Ad-SP/PR39的细胞显示出了更强的抗菌活性[BCG活菌数分别为(8.5±1.4)×106、(6.9±1.8)×106cfu/ml,P<0.05)],且能有效吞噬并杀灭胞内菌。结论本课题组构建的重组腺病毒能够靶向巨噬细胞表达抗菌肽PR39基因,发挥抗胞内菌作用,为抗菌肽在胞内菌感染治疗中的应用开辟了新思路。
Objective To detect the antibacterial activity of recombinant adenovirus Ad-SP/PR39 target expressing PR39 in mouse macrophages. Methods The recombinant adenovirus particles Ad-SP/PR39 were purified with cesium chloride density gradient centrifugation. The expression of green fluorescent protein （GFP） was identified,and the titer of virus was detected by fluorescent microscopy. The purified recombinant adenovirus was then used to infect mouse macrophage RAW264.7 cells,human colon carcinoma Lovo cells,human hepatocellular carcinoma HepG2 cells,human breast carcinoma ZR-75-30 cells and African green monkey kidney COS-7 cells. RT-PCR and immunocytochemistry were employed to detect the expression of PR39 gene in these cell lines. The lysate of macrophage RAW264.7 cells infected by Ad-SP/PR39 was added into medium of Bacille de Calmette-Guerin （BCG） to detect its antibacterial activities in vitro. The RAW264.7 cells were allowed to engulf BCG,and then infected by Ad-SP/PR39 before counting the number of living bacteria in RAW264.7 cells to detect the intracellular antibacterial activity. Results After purification and concentration,the titer of the recombinant adenovirus reached 5×10 11 efu/ml,which could infect many cell strains such as RAW264.7 cell efficiently. RT-PCR and immunocytochemistry showed that the antimicrobial peptides PR39 gene expressed specifically in mouse macrophage RAW264.7,while hardly expressed in other cell lines. Compared with the RAW264.7 cells infected with Ad-C,the RAW264.7 cells infected with Ad-SP/PR39 showed stronger antibacterial ability in vitro [the number of viable BCG were （8.5±1.4）×106 vs （6.9±1.8）×106cfu/ml,respectively,P0.05]. The RAW264.7 cells infected with Ad-SP/PR39 could engulf and kill BCG efficiently. Conclusions The recombinant adenovirus Ad-PR39 can express antimicrobial peptide PR39 in mouse macrophage RAW264.7 cells and enhance its antimicrobial ability. The present study has provided a foundation of new gene therapy for intracellular bacterial infection with antimicrobial peptides.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2010年第8期993-996,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金(30600790)
关键词
基因表达
腺病毒科
巨噬细胞
agene expression
adenoviridae
macrophages
