TIM2基因修饰对H22细胞的抑制作用及苦参碱的增强作用

Enhancing effect of matrine on the tumor-inhibition by TIM2 gene-modified hepatocarcinoma H22 cells in mice

目的观察苦参碱对TIM2基因修饰小鼠H22肝癌细胞瘤苗的体内作用。方法以携带小鼠TIM2基因的真核表达载体脂质体法转染H22细胞，经体外稳定筛选获得TIM2转基因H22细胞瘤苗。建立小鼠肝癌移植瘤模型，接种TIM2转基因H22细胞瘤苗（H22-TIM2组），观察成瘤情况，并设空载体转染的H22细胞稳定表达株和H22细胞作为对照（H22-EGFP组和H22组）；同时，分别加用药物苦参碱治疗，观察苦参碱对不同处理组小鼠肿瘤生长情况的影响。结果成功得到TIM2转基因修饰H22细胞，鉴定有TIM2基因mRNA及EGFP的稳定表达。免疫接种小鼠后，在小鼠体内的成瘤率为41．6％，明显低于H22组和H22-EGFP组（后两者成瘤率在91．6％以上）（P〈0．01），小鼠肿瘤的生长速率也较其他各组明显缓慢，实验结束时H22-TIM2组小鼠肿瘤平均体积为（31．344-9．21）mm^3，明显小于H22-EGFP组和H22组[（98．25±25．23）mm^3和（114．08±36．45）mm^3；P〈0．01]。接种H22-TIM2细胞对小鼠肿瘤的抑制率为69．2％，加用苦参碱后，抑瘤率达90．6％，明显高于单用苦参碱组（67．5％）及H22-EGFP和苦参碱联用组（70．8％）。结论TIM2基因修饰H22细胞可显著降低H22肝癌细胞在小鼠体内的致瘤性，抑制小鼠体内H22移植瘤的发生和发展，而苦参碱可明显改善其体内抗癌活性。

Objective To investigate the effects of matrine on the anti-tumor efficiency of TIM2 gene-modified murine hepatocarcinoma H22 cells. Methods A combined eukaryotic expression vector pIRES2-EGFP-TIM2 was constructed and transfected into H22 cells by lipofectamin. The monoclone of positive H22-TIM2 cells and negative control H22-EGFP cells transfected with pIRES2-EGFP vector were selected by G418 pressure and limited dilution method in turn and were inoculated to establish the tumorbearing mouse model. Next, matrine was administered to the tumor-bearing mice and the inhibitory effect of matrine was determined. Results The co-expression of EGFP protein and TIM2 gene was detected in H22 cells selected after TIM2 gene transfecion. After subcutaneous injection of H22-TIM2 cells, the rate of tumor formation （41%） was lower than that of H22 cells and H22-EGFP cells injection （92%） in mice. The tumor growth was significantly inhibited in mice vaccinated with H22-TIM2 cells. After the experiment was completed, the volume of tumors in mice of H22-TIM2 group was 31.34±9.21 mm^3 , smaller than those in H22-EGFP group （ 98.25 ± 25.23 ） mm^3 and H22 cells group （ 114.08 ± 36.45 ）mm^3 （ P 〈 0.01 ）. Matrine dramatically enhanced the anti-tumor efficiency of TIM2 gene-modified H22 cells, with the highest tumor inhibitory rate （IR） 90.6% among the H22-TIM2 group, matrine treatment group and H22-EGFP cells combined with matrine treatment group （69.2% , 67.5% and 70.8% , respectively ） in the experimental mice. Conclusion The tumorigenesity of H22 cells has been markedly impaired after modification by TIM2 gene. Matrine can enhance its inhibitory effect on tumors of H22-TIM2 cells in vivo. These data indicate importance to further study on the biological role of TIM2 gene in tumor immunity and explore the molecular mechanism of matrine in suppressing of tumor growth.