小鼠肠癌RNA转染mIL-12修饰的树突细胞诱发特异性细胞毒性T淋巴细胞的研究

Specific activity of cytotoxic T lymphocyte in mice induced by dendritic cells after mIL-12 gene modification and murine colon carcinoma RNA transfection

目的:研究小鼠结肠癌细胞CT-26 RNA体外转染mIL-12基因修饰的树突细胞(DC),观察其诱导特异性细胞毒性T淋巴细胞(CTL)能力。方法:小鼠骨髓细胞体外经rmGM-CSF、rmIL-4诱导培养获取DC,流式细胞仪检测其纯度;293细胞扩增携带mIL-12基因的重组腺病毒,体外转染DC;Trizol法提取CT-26细胞总RNA,应用TransM essenger体外转染mIL-12基因修饰的DC;ELISA法检测细胞上清及小鼠血液中mIL-12,LDH释放法检测小鼠体内特异性CTL活性。结果:小鼠骨髓细胞经诱导培养后,获得大量高纯度的DC,流式细胞仪检测CD11 c+的DC>90%;携带mIL-12基因重组腺病毒转染的DC细胞高表达mIL-12;CT-26细胞总RNA体外转染mIL-12基因修饰的DC后,回输小鼠,能明显提高小鼠体内mIL-12的水平,并可诱导体内生成较高水平的CTL活性,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,可诱导机体生成中等水平的特异性CTL活性,DC、PBS对照组则均无此作用。结论:小鼠肠癌CT-26细胞总RNA转染mIL-12基因修饰的DC后,免疫接种小鼠,可提高小鼠体内m IL-12的水平,并能有效诱导机体产生较高水平的特异性CTL活性。

Objective：To investigate specific activity of cytotoxic T lymphocyte （CTL） induced by mIL-12 gene modified dendritic cells （DC） transfected with the total RNA of CT-26 （a cell line of murine carcinoma of colon）. Methods ： In vitro proliferation of DC from the culture of murine bone marrow was stimulated by rmGM-CSF and rmIL-4, and the purity of DC was detected by flow-cytometry. The generated DC were then modified by adenovirus with mIL-12 gene which were proliferated in 293 cells.The total RNA of CT-26 was obtained through Trizol＇s process, and transfected into the mIL-12 gene modified DC by TransMessenger in vitro. The levels of mIL-12 both in vitro and in vivo and the activity of CTL in vivo were estimated with enzyme linked immunosorbent assay and modified lactate dehydrogenase release assay respectively. Results：Plenty of DCs were obtained from the culture of murine bone marrow, with over 90 % of CD11c^＋ cells. DC modified by mIL-12 gene could induce high level of mIL-12 both in vitro and in vivo, and the differences compared to control groups were significant （P 〈 0.01 ）. Vaccination with mIL-12 gene modified DC transfected with the total colon carcinoma RNA could induce high level of specific CTL activity in vivo, and the Ad-LacZ modified DC and DC transfected with the same total RNA induced moderate level of specific CTL activity in vivo, meanwhile the level of specific CTL activity was quite low in the mice immunized with DC or phosphate buffer. Conclusion：mIL-12 gene modified DC transfected with the total RNA of tumor could raise the level of mIL-12 in mice, and could also activate specific CTL effectively.