反义IDO-AFP基因融合修饰树突状细胞抗肝癌免疫作用

Antitumor effect of dendritic cells modified by antisense-IDO-AFP with controling tryptophan metabolism

目的探讨反义IDO-AFP基因融合修饰树突状细胞(DC)后,DC通过调控色氨酸代谢达到高效、特异性抗肝癌作用。方法采用细胞培养方法,从小鼠骨髓中获得DC,用反义IDO核酸和AFP基因融合的载体,转染入DC内,检测转染入融合基因DC的对T细胞增殖的影响和体外对肝细胞癌的免疫应答的改变。以^(51)Cr释放法测定细胞毒性T淋巴细胞(CTL)杀伤活性。制作小鼠肝癌模型,分别予以反义IDO-AFP修饰的DC、单纯DC、空白对照组进行治疗,检测血清中自细胞介素(IL)-10、干扰素(IFN)-7水平；CD4、CD8和IFN-7和IL-10双阳性细胞比例,观察其抑制肿瘤的效果。结果反义IDO-AFP修饰的DC可激活小鼠脾淋巴细胞；能对肝癌细胞株H22细胞进行特异性杀伤,杀伤率为89．3%,显著高于单纯DC、生理盐水(空白对照组)组的34．7%和8．9% (P均＜0．01)而对艾氏腹水瘤细胞无效；反义IDO-AFP修饰DC组色氨酸含量为(17．8±1．2)μm,而单纯DC组和空白对照组的色氨酸为(6．2±0．6),um和(5．2±1．2)μm,表明反义IDO-AFP修饰DC可明显增加培养液中的色氨酸。使Thl细胞比例上升,抑制体内肿瘤的生长。结论反义IDO-AFP基因融合修饰树突状细胞后,DC通过调控色氨酸代谢达到高效、特异性抗肝癌的目的,可成为一种有效的瘤苗。

Objective To investigate the effects of antitumor effect of dendritic cells modified by antisense-IDO-AFP with controling tryptophan metabolism in vitro and in vivo. Methods DCs were cultured from bone marrow ceLLs and modified by antisense-IDO-AFP. Spleen lymphocytes of mice were activated by modified DCs and the cytotoxicity was detected by ^51Cr release method. Modified DCs, pure DCs and control DCs were injected into mice bearing H22 liver cancer cells to observe the levels of IL-10, IFN- γ in serum and the alteration of proportions of CD8 ^＋ -IFN-γ^＋ and CD8 ^＋ -IL-10 ^＋ cells, CD4^ ＋-IFN-γ^＋ and CD4^ ＋ -IL-10^ ＋ cells. Results DCs modified by antisense-IDO-AFP could activate spleen lymphocytes and the latter could specifically kill H22 cells, and the lyse rate to H22 cancer cells was 89.3 %, but not to Ehrilich ascite carcinoma cells. Modified DC could improve the host＇ s antitumor effect of DCs modified by antisense-IDO-AFP with controling tryptophan metabolism in vitro and in vivo, and the increased proportions of Thl cells could inhibit tumor growth. Conclusion DCs transfected with antisense-IDO-AFP gene could induce specific cytotoxic T lymphocytes and strikingly raise DCs＇ antigen present function, and have specific CTL killing activity.