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目的:研究以小鼠结肠癌细胞CT-26RNA作为抗原体外转染经mIL-12基因修饰的树突状细胞(dendriticcells,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rmGM-CSF、rmIL-4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mIL-12基因的重组腺病毒,体外转染树突状细胞;Trizol法提取CT-26细胞总RNA,应用Trans-Messenger体外转染mIL-12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mIL-12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CT-26细胞总RNA体外经TransMessenger介导,转染mIL-12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Ad-LacZ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mIL-12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。
OBJECTIVE: To study the effect of mIL-12 gene-modified dendritic cells (DC) transfected with CT-26RNA of mouse colon cancer cells as antigen in vitro to observe its specific anti-tumor effect. Methods: The dendritic cells were induced by rmGM-CSF and rmIL-4 in vitro. The purity of the dendritic cells was detected by flow cytometry. The recombinant adenovirus carrying mIL-12 gene was amplified by 293 cells and transfected into dendritic The Trizol method was used to extract the total RNA of CT-26 cells. The mIL-12 gene-modified dendritic cells were transfected with Trans-Messenger in vitro and the mice were immunized. The levels of mIL-12 in the supernatant and the blood of the mice were detected by ELISA, and cytotoxic T lymphocyte (CTL) cytotoxic activity was detected by LDH release assay. Results: A large number of high purity dendritic cells were obtained after induction of bone marrow cells in mice. Flow cytometry was used to detect CD11c + dendritic cells> 90%. Total RNA extracted from CT-26 cells was transduced with TransMessenger in vitro, After transfection with mIL-12 gene-modified dendritic cells, the transfection of mice can induce a higher level of specific CTL activity in vivo. After the parental tumor inoculation, mice are 100% (P <0.01). After the tumor inoculation, the survival rate of 60% of the mice in the control group and DC transfected with RNA were significantly lower than those in the experimental group The control group did not induce the body to produce specific CTL activity, mice did not long-term survival. CONCLUSION: Dendritic cells transfected with mouse CT26 cells transfected with RNA and mIL-12 gene can effectively induce tumor antigens in vivo and induce high levels of CTL in the body and induce more specific Anti-tumor effect.