研究以小鼠结肠癌细胞CT26 RNA作为抗原体外转染经mIL12基因修饰的树突状细胞（dendritic cells，DC），观察其诱导特异性抗肿瘤的效应。 方法：小鼠骨髓细胞体外以rmGMCSF、rmIL4诱导培养获取树突状细胞，流式细胞术检测纯度；293细胞扩增携带mIL12基因的重组腺病毒，体外转染树突状细胞；Trizol法提取CT26细胞总RNA，应用TransMessenger体外转染mIL12基因修饰的树突状细胞，免疫接种小鼠。 ELISA法检测细胞上清及小鼠血液中mIL12水平，LDH释放法检测小鼠体内细胞毒性T淋巴细胞（CTL）杀伤活性。结果：小鼠骨髓细胞经诱导培养后，获得大量高纯度的树突状细胞，流式细胞术检测CD11c+的树突状细胞> 90%；提取的CT26细胞总RNA体外经TransMessenger介导，转染mIL12基因修饰的树突状细胞后，回输小鼠，可以诱导体内生成较高水平的特异性CTL活性，亲本肿瘤接种后小鼠100%长期存活，而以该RNA转染AdLacZ修饰DC后的对照组及RNA转染DC的对照组，诱导机体生成的特异性CTL活性显著低于实验组（P<0.01），亲本肿瘤接种后小鼠60%长期存活，DC、PBS对照组则均未诱导机体生成特异性CTL活性，小鼠无长期存活。结论： 树突状细胞经小鼠结肠癌CT26细胞RNA转染和mIL12基因修饰后免疫接种小鼠，可在体内有效提呈肿瘤抗原，诱导机体产生高水平的CTL，更有效地诱发特异性抗肿瘤效应。

To investigate the specific antitumor effect of mIL12 gene modified dendritic cells (DC) transfected with the total RNA of CT26 murine colon carcinoma. Methods: DC were cultured from murine bone marrow in the presence of rmGMCSF and rmIL4.The purity of DC was detected by flowcytometry. Adenovirus carrying mIL12 gene was proliferated in 293 cells and was transfected into DC. The total RNA of CT26 was extracted by Trizol reagent and was introduced into mIL12 gene modified DC by TransMessenger in vitro. The modified DC were used to immunize mice. The in vitro and in vivo levels of mIL12 and the in vivo activity of cytotoxic T lymphocyte（CTL）were examined by ELISA assay and modified LDH release assay, respectively. The tumor growth and animal survival time of immunized mice were estimated after they were challenged with parental tumor cells. Results: DC were successfully obtained from the culture of murine bone marrow, with CD11c+ accounting for over 90%. Vaccination with mIL12 gene modified DC transfected with the total RNA of CT26 induced strong specific CTL activity in vitro. All the immunized mice survived for a long term. Vaccination with AdLacZ modified DC and DC transfected with the total RNA induced moderate specific CTL activity in vitro（P<0.01）, and 60% of the immunized mice survived for a long time. There was no specific CTL activity in the mice immunized with DC and PBS and all the mice died. Conclusion: mIL12 gene modified DC transfected with the total RNA of CT26 can effectively present endogenetic tumor antigen and induce strong CTL activity, thus inducing more specific antitumor immune response.

R730.3

江苏省“六大人才高峰”资助项目（No. 2005A4）; 南京军区南京总医院院管基金重点资助项目（No. 2006006）