肝癌细胞特异性IL-1β反义RNA对小鼠移植肝癌的抑制  

Hepatocarcinoma specific IL-1β anti-sense RNA inhibits implanted hepatocarcinoma in mice

作　　者：刘艳艳[1] 梁淑娟[1] 王焕芹[1] 张素华[1] 肖伟玲[1] 吴慧娜[1] 

机构地区：[1]潍坊医学院免疫学教研室,山东省高校免疫学重点实验室,山东潍坊261042

出　　处：《中国肿瘤生物治疗杂志》2009年第3期253-257,共5页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.30772497);山东省高层次卫生人才1020工程专项基金;教育部科学技术研究基金重点项目(No.205090);山东省科技攻关计划项目(No.2008GG10002007)~~

摘　　要：目的:构建肝癌细胞特异性小鼠IL-1β反义RNA表达载体,观察其对H22肝癌细胞小鼠移植瘤生长的影响及其相关机制。方法:构建由AFP最小启动子和CMV增强子嵌合序列调控的携IL-1β反义RNA表达载体pafpIRES2-antiIL-1β1和pafpIRES2-antiIL-1β2,经质粒PCR、限制性酶谱分析、序列测定进行鉴定。反义RNA表达载体转染小鼠H22肝癌细胞,分H22/mock组、H22/antiIL-1β1组、H22/antiIL-1β2三组,RT-PCR检测IL-1β的表达水平。以转染后的H22细胞皮下接种建立荷肝癌小鼠模型,观察移植瘤体积和重量,MTT法检测荷瘤小鼠脾脏中分离的NK细胞对H22细胞的杀伤活性。结果:经质粒PCR、限制性酶谱分析、序列测定证实成功构建能够在肝癌细胞中特异性表达IL-1β反义RNA的表达载体pafpIRES2-anti-IL-1β1和pafpIRES2-antiIL-1β2,转染H22细胞后细胞中IL-1β表达水平明显下降,以pafpIRES2-antiIL-1β2更为显著。成功建立荷肝癌小鼠模型,与H22/mock组小鼠相比,H22/antiIL-1β2组小鼠移植瘤体积较小,生长显著减慢(P<0.05)。H22/anti-IL-1β1、H22/antiIL-1β2组荷瘤小鼠的NK细胞对H22细胞的杀伤活性明显增强(P<0.05或P<0.01)。结论:成功构建的肝癌细胞特异性IL-1β反义RNA表达载体可有效抑制小鼠移植肝癌的生长,其机制与靶向阻断IL-1β表达、上调NK细胞的杀伤活性有关。Objective ：To construct hepatocarcinoma specific IL-1β anti-sense RNA expression vector and to explore its effect on the growth of implanted hepatocarcinoma H22 cells in mice and the possible mechanism. Methods： Murine IL- l β anti-sense RNA expression vectors pafplRES2-antiIL-1β1 and pafpIRES2-antiIL-1β2 under the regulation of minimal alpha-feto protein （AFP） promoter and CMV enhancer were constructed, and further verified by PCR, restriction endonu- clease analysis and DNA sequencing. H22 cells transfected with pafpIRES2-antiIL-1 β 1 or pafpIRES2-antiIL-1 β 2 were divided into 3 groups ： H22/mock, H22/antilL-1 β1 and H22/antiIL-1β2 group. Expression of IL-1β was detected by RT- PCR. Transfected H22 cells were subcutaneously injected into mice to establish tumor implanted mouse model. Tumor volume was measured; the cytotocixity of spleen NK against H22 cells was detected by MTr. Results： Hepatocarcinoma specific IL-1 β anti-sense RNA expression vectors pafplRES2-antilL-1 β and pafplRES2-antiIL-1 β2 were successfully constructed and were verified by PCR, restriction endonuclease analysis and DNA sequencing. IL-1β expression in H22 cells was down-regulated after transfected with IL-1β~ anti-sense RNA expression vectors, especially with the pafpIRES2-antiIL- 1 β2 vector. Hepatocarcinoma cells implanted mouse model was successfully established. Tumor volume and growth of tumor in H22/antilL-1β2 mice was obviously smaller than that in H22/mock mice, and the cytotocixity of spleen NK against H22 cells in H22/antilL-1β1 and H22/antilL-1β2 mice was also greatly enhanced. Conclusion： Hepatoearcinoma specific IL-113 anti-sense RNA expression vector pafplRES2-antilL-1β was successfully constructed. It effectively inhibits the growth of implanted hepatocarcinoma in mice probably through specifically blocking expression of IL-1β and increasing cytotocixity of spleen NK.

关 键 词：肝癌 白细胞介素1Β 反义RNA NK细胞 

分 类 号：R735.7[医药卫生—肿瘤] R735.54[医药卫生—临床医学]