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。〖HT5W〗结果: 〖HT5"SS〗 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antiIL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/antiIL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1β antisense RNA expression vectors pafpIRES2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 endonuclease analysis and DNA sequencing. H22 cells transfected with pafpIRES2antiIL1β 1 or pafpIRES2antiIL1β 2 were divided into 3 groups: H22/mock, H22/antiIL1β1 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 MTT. Results: Hepatocarcinoma specific IL1β antisense RNA expression vectors pafpIRES2antiIL1β1 and pafpIRES2antiIL1β2 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1β2 vector. Hepatocarcinoma cells implanted mouse model was successfully established. Tumor volume and growth of tumor in H22/antiIL1β2 mice was obviously smaller than that in H22/mock mice, and the cytotocixity of spleen NK against H22 cells in H22/antiIL1β1 and H22/antiIL1β2 mice was also greatly enhanced. Conclusion: Hepatocarcinoma specific IL1β antisense RNA expression vector pafpIRES2antiIL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.

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)