可调控慢病毒介导白细胞介素-12治疗结肠癌的研究  

Treatment of colon cancer by regulatable lentivirus-mediated interleukin-12 gene

作　　者：刘业六[1] 张逖[1] 杨晓钟[2] 戴伟杰[2] 张娟[2] 唐晓军[1] 

机构地区：[1]南京医科大学附属淮安第一医院胃肠外科,淮安223300 [2]南京医科大学附属淮安第一医院消化内科,淮安223300

出　　处：《中华实验外科杂志》2014年第8期1749-1751,共3页Chinese Journal of Experimental Surgery

基　　金：南京医科大学科技发展基金重点项目（09NJMUZ30）

摘　　要：目的 观察RU486可调控慢病毒介导的小鼠白细胞介素-12（mIL-12）基因对小鼠结肠癌C26细胞移植瘤生长和微血管形成的影响.方法 应用分子同源重组技术构建、包装能表达mIL-12的RU486可调控慢病毒LV-RUmIL-12.将LV-RUmIL-12重组慢病毒感染C26细胞,酶联免疫吸附试验（ELISA）法检测不同浓度RU486调控下mIL-12在C26细胞中的表达.构建小鼠结肠癌C26细胞皮下移植瘤模型,将100只小鼠分为5组[磷酸盐缓冲液组（PBS组）、空白慢病毒组（LV组）、RU486组、LV-RUmIL-12组和LV-RUmIL-12-RU486组],观察小鼠肿瘤生长情况.21 d后处死小鼠,检测小鼠血清mIL-12水平,摘除瘤体,称瘤重,免疫组织化学方法检测肿瘤微血管密度（MVD）和淋巴细胞浸润情况.结果 成功构建、包装RU486可调控慢病毒LV-RUmIL-12.LV-RUmIL-12慢病毒感染C26细胞后,1×10-5 mol/L的RU486可诱导1×106个细胞产生（148±25） μg/L的mIL-12蛋白.LV-RUmIL-12-RU486、PBS、LV、RU486、LV-RUmIL-12组小鼠血清mIL-12水平分别为（3 572.60±245.60）、（145.43±13.53）、（134.53±12.86）、（147.32±15.56）和（139.67±14.84） μg/L,LV-RUmIL-12-RU486组mIL-12水平显著增高（P＜0.05）.RU486诱导下的LV-RUmIL-12慢病毒能够抑制小鼠C26细胞移植瘤的生长,抑瘤率达72.59％;LV-RUmIL-12-RU486组移植瘤组织MVD（12.5±4.3）少于PBS （45.5±5.7）、LV （39.5±3.7）、RU486（42.4±7.5）和LV-RUmIL-12组（47.2±2.6）,差异有统计学意义（P＜0.05）;LV-RUmIL-12-RU486组肿瘤组织中CD4＋和CD8＋淋巴细胞浸润较对照组增多,差异有统计学意义（P＜0.05）.结论 携带mIL-12基因的RU486可诱导慢病毒能够有效抑制小鼠结肠癌皮下移植瘤微血管生成,对结肠癌的生长有明显的抑制作用.Objective To evaluate the efficiency of interleukin-12 （IL-12） gene mediated by RU486 regulatable lentivirus system on tumor growth and microvessel angiogenesis of mouse colon cancer C26 cells subcutaneously implanted tumor.Methods The mouse IL-12 gene was recombined to construct the RU486 regulatable lentivirus transfer plasmid PNL-LV-RUmIL-12.The LV-RUmIL-12 lentiviruses were produced by co-transfecting PNL-LV-RUmIL-12.The expression of IL-12 in C26 colon cancer cells infected by RU486 regulated LV-RUmIL-12 lentivirus was detected by enzyme linked immunosorbent assay （ELISA） kit.Colon cancer model was established by subcutaneously inoculating C26 cells into the BALB/c mice.One hundred mice were divided into 5 groups：PBS group,blank lentivirus group （LV group）,RU486 group,LV-RUmIL-12 group and LV-RUmIL-12-RU486 group.The tumor size changes were calculated.After treatment for 21 days,the mice were sacrificed by cervical dislocation.Serum levels of IL-12 were assayed by enzyme linked immunosorbent assay （ELISA）.Immunohistochemical staining was employed to determine microvessel density （MVD） and CD4 ＋ and CD8 ＋ lymphocytes in the tumor tissues.Results After injection of LV-RUmIL-12 lentivirus,the expression of IL-12 protein in C26 cells was RU486 dose-dependent.The high-est expression level of IL-12 protein in 1 × 106 C26 cells induced by 1 × 10-5 mol/L RU486 was （148 ± 25） μg/L.Serum levels of IL-12 were （3 572.60 ±245.60）,（145.43 ± 13.53）,（134.53 ± 12.86）,（147.32 ± 15.56） and （139.67 ± 14.84） μg/L in LV-RUmIL-12-RU486,PBS,LV,RU486 and LV-RUmIL-12 groups respectively,and IL-12 level was significantly increased in LV-RUmIL-12-RU486 group （P ＜ 0.05）.The tumor growth in LV-RUmIL-12-RU486 group was markedly inhibited.The inhibition rate in LV-RUmIL-12-RU486 group was 72.59％.The MVD in LV-RUmIL-12-RU486 group （12.5 ± 4.3） was statistically decreased as copared with PBS group （45.5 ±5.7）,LV group （39.5 ± 3.7）,RU486 group （42.4 ±7.5）

关 键 词：白细胞介素-12 结肠癌 慢病毒 血管生成 

分 类 号：R735.35[医药卫生—肿瘤]