摘要
目的:制备载胞苷脱氨酶-胸腺嘧啶激酶(colicytocinedeaminase-thymidinekinase,CD-TK)双自杀基因的超声微泡,检测其对肺癌的杀伤效应。方法:制备载CD-TK双自杀基因的微泡,观察形态和大小,用碘化丙啶染质粒,观察荧光和计算微泡与基因的结合率。培养肺癌H1299细胞系和建立肺癌裸鼠模型,随机分为对照、超声(ultrasound,U)+微泡(microbubble,M)+CD-TK、U+M+CD-TK+5-FC(5-fluorcytosine,5-氟胞嘧啶)、U+M+CD-TK+GCV(ganciclovir,丙氧鸟苷)、U+M+CD-TK+5-FC+GCV 5组,每组1×107个细胞。微泡转染H1299肺癌细胞,24 h后观察细胞绿色荧光蛋白(green fluorescent protein,GFP)荧光表达情况,测定细胞GFP转染效果和每组24 h细胞凋亡和周期。体内动物实验,每组6只裸鼠,对照组注射200μL PBS,其余各组尾静脉注射等量的载CD-TK双自杀基因的微泡。观察微泡的显影,同时在肿瘤部位超声辐照转染基因,每组按实验分组连续腹腔注射5-FC、GCV一周,每周记录每只裸鼠肿瘤的最长径和最短径,计算并记录肿瘤的体积变化。第5周取裸鼠肿瘤标本。免疫荧光测TdT(TUNEL法)和PCNA的表达,评估肿瘤组织细胞凋亡和增殖情况。结果:成功制备出载CD-TK双自杀基因的微泡,无毒前药5-FC和GCV联合载CD-TK双自杀基因的超声微泡治疗效果明显,有促进凋亡[凋亡率(28.45±1.75)%]、抑制增殖作用[S期(61.40±4.31)%],疗效明显优于对照组及单药组(5-FC组P=0.002和GCV组P=0.012,对照组P=0.000)。双药联合超声辐照载基因微泡组的肿瘤体积最小、质量最小、凋亡细胞最多、增殖受抑制最明显。结论:无毒前药5-FC+GCV联合微泡载CD-TK双自杀基因能促进肺癌细胞凋亡,抑制其增殖,对肺癌有明显杀伤作用。
Objective:To prepare the ultrasound microbubbles loaded with the cytidine deaminase-thymidine kinase(CD-TK)double suicide gene,and to investigate its killing effect on lung cancer. Methods:The microbubbles loaded with the CD-TK double suicide gene were prepared,and their morphology and size were observed. Propidium iodide was used for the staining of plasmids to observe fluorescence and calculate the binding rate of microbubbles and genes. Lung cancer H1299 cells were cultured and a nude mouse model of lung cancer was established. The cells were randomly divided into control group,ultrasound(U)+microbubble(M)+CD-TK group,U+M+CD-TK+5-fluorcytosine(5-FC)group,U+M+CD-TK+ganciclovir(GCV)group,and U+M+CD-TK+5-FC+GCV group,with 1×107 cells in each group. The lung cancer H1299 cells were transfected with the microbubbles,and after 24 hours,the expression of green fluorescent protein(GFP)was observed and the effect of GFP transfection and 24-hour apoptosis and cell cycle were measured.In the animal experiment in vivo,there were 6 nude mice in each group;the mice in the control group were treated with the injection of 200 μL phosphate-buffered saline,and those in all the other groups were given the tail vein injection of an equal volume of the microbubbles loaded with the CD-TK double suicide gene. The development of microbubbles was observed,and gene transfection was performed at the tumor site by ultrasound irradiation.Intraperitoneal injection of 5-FC and GCV was performed for one week. The longest and shortest diameters of the tumors of each nude mouse were recorded every week,and the change in tumor volume was calculated and recorded. Tumor samples were collected for the nude mice at week 5;immunofluorescence assay was used to measure the expression of TdT(the TUNEL method)and proliferating cell nuclear antigen(PCNA),and cell apoptosis and proliferation in tumor tissue were assessed. Results:The microbubbles loaded with the CD-TK double suicide gene were successfully prepared. 5-FC and GCV combined with ultrasound microbubbles loaded with CD-TK double suicide gene had a marked therapeutic effect and significantly promoted apoptosis[an apoptosis rate of(28.45±1.75)%] and inhibited proliferation[(61.40±4.31)% cells in S phase),with a significantly better effect than the control group(P=0.000),the 5-FC group(P=0.002),and the GCV group(P=0.012). The U+M+CD-TK+5-FC+GCV group had the lowest tumor volume and weight,the highest number of apoptotic cells,and the most significant inhibition of proliferation. Conclusion:5-FC and GCV combined with ultrasound microbubbles loaded with the CD-TK double suicide gene can promote the apoptosis and inhibit the proliferation of lung cancer cells and thus have a marked killing effect on lung cancer.
作者
胡聪
杜晓燚
江德鹏
杨旭
张荣贵
Hu Cong;Du Xiaoyi;Jiang Depeng;Yang Xu;Zhang Ronggui(Department of Respiratory Medicine,the Second Affiliated Hospital,Chongqing Medical University;Department of Urinary Surgery, the Second Affiliated Hospital,Chongqing Medical University)
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2019年第2期157-162,共6页
Journal of Chongqing Medical University
基金
国家自然科学基金资助项目(编号:81650003)
重庆市科委资助项目(编号:cstc2015jcyjA10030)
关键词
肺癌
自杀基因
微泡
超声
lung cancer
suicide gene
microbubble
ultrasound
