CAJ | 학술논문

目的研究IL-24基因修饰的树突状细胞(DC)与同源细胞因子诱导的杀伤细胞(cyto-kine-induced killer,CIK)共培养后对A549肺癌细胞的杀伤作用及其机制.方法从健康人外周血单个核细胞中常规诱导DC、CIK细胞,同时抽提重组腺病毒质粒pAdEasy-1-pTrack-CMV-IL-24,Pac I酶切线性化后脂质体转染QBl-293A细胞,收获Ad-IL-24重组病毒.将IL-24基因通过重组病毒导入已负载肿瘤抗原的DC,获得细胞称为DC-IL-24.RT-PCR和ELISA法检测DC中IL-24基因的表达,FCM和ELISA法检测DC表型及分泌细胞因子能力的变化,将DC和CIK细胞混合培养,溶血试验检测CIK细胞产生穿孔素的能力,FCM法检测共培养的DC-CIK细胞对A549肺癌细胞细胞毒活性的变化.结果获得了高滴度的重组病毒Ad-IL-24并成功将IL-24基因导入DC,在倒置荧光显微镜下可观察到荧光,IL-24可上调Dc表而CD80、CD83、HLA-DR、CD40、CXCR4分子的表达,转染后Dc分泌IL-12、TNF-α和IL-24的能力显著增强,DC-IL-24更能促进CIK细胞产生穿孔素,与同源CIK细胞共培养后对A549肺癌细胞的细胞毒活件明显增强.结论 IL-24基因修饰的DC能增强自体CIK细胞产生特异性抗肿瘤免疫,其机制与IL-24能促进DC表型成熟,分泌Th1型细胞因子,维持DC活化状态,进而促进CIK细胞活化密切相关.
목적 연구IL-24기인수식적수돌상세포(DC)여동원세포인자유도적살상세포(cyto-kine-induced killer,CIK)공배양후대A549폐암세포적살상작용급기궤제.방법 종건강인외주혈단개핵세포중상규유도DC、CIK세포,동시추제중조선병독질립pAdEasy-1-pTrack-CMV-IL-24,Pac I매절선성화후지질체전염QBl-293A세포,수획Ad-IL-24중조병독.장IL-24기인통과중조병독도입이부재종류항원적DC,획득세포칭위DC-IL-24.RT-PCR화ELISA법검측DC중IL-24기인적표체,FCM화ELISA법검측DC표형급분비세포인자능력적변화,장DC화CIK세포혼합배양,용혈시험검측CIK세포산생천공소적능력,FCM법검측공배양적DC-CIK세포대A549폐암세포세포독활성적변화.결과 획득료고적도적중조병독Ad-IL-24병성공장IL-24기인도입DC,재도치형광현미경하가관찰도형광,IL-24가상조Dc표이CD80、CD83、HLA-DR、CD40、CXCR4분자적표체,전염후Dc분비IL-12、TNF-α화IL-24적능력현저증강,DC-IL-24경능촉진CIK세포산생천공소,여동원CIK세포공배양후대A549폐암세포적세포독활건명현증강.결론 IL-24기인수식적DC능증강자체CIK세포산생특이성항종류면역,기궤제여IL-24능촉진DC표형성숙,분비Th1형세포인자,유지DC활화상태,진이촉진CIK세포활화밀절상관.
Objective To study the antitumor effect and mechanism of co-cultured cytokine-induced killer(CIK) cells and autologous DC modified with IL-24 gene on A549 cells in vitro. Methods DC and CIK cells were prepared routinely from human peripheral blood mononuclear cells(PBMC). Recombinant adenovirus vector pAdEasy-1-pTrack-CMV-IL-24 was extracted from DH5α, it was lineared with Pac I and transfected into A293 cells, and then the IL-24 recombined adenovirus(Ad-IL-24) was obtained. Ad-IL-24 was used to infect DC. The cells obtained were named DC-IL-24. RT-PCR and ELISA were used to evaluate the expression of IL-24 gene in transfected DC. The phenotypes change of DC were identified by flow cytometry analysis, the concen-tration of IL-12 and TNF-α in supernatant of DC were determined by EIJSA. The ability of CIK producing per-forin was measured by homolysis method. FCM was used to determine the cytotoxicity of cocultured CIK cells and autologous DC modified with IL-24 gene to A549 cells. Results We obtained the high titre of Ad-IL-24.IL-24 gene was transfered into DC successfully via Ad-IL-24. The green fluorescence was observed on DC by fluorescence microscope. The expression rate of CD80, CD83, HI.A-DR, CD40, CXCR4 on DC-IL-24 was sig-nificantly increased compared with that of the control group. DC-IL-24 produced markedly higher levels of IL-12 and TNF-α as compared with DC. DC-IL-24 can enhance the ability of CIK cells producing perforin. On com-parison with non-transfected DC co-cultured with CIK cells, transfected DC co-cultured with CIK cells had a sig-nificantly higher lytic activity against A549 cells. Conclusion IL-24 gene modification can enhance the anti-tu-moral immunity of DC. The mechanism of which might be related to the increased secretion of IL-12 and TNF-α, up-regulation expression of co-stimulatory molecules and MHC Ⅱ class molecules on DC, promoting the acti-vation and maturation of DC, and then enhancing CIK cells to generate specific anti-tumoral immunity.