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目的探讨逆转录病毒携带白细胞介素(IL)12转染树突状细胞(DC)体外诱导免疫杀伤肝癌细胞的效能及其机制。方法感染指数(MOI)为100,含IL12的重组逆转录病毒修饰肝癌患者外周血DC(DCIL12),酶联免疫吸附试验法(ELISA)检测DCIL12(5×103个/ml)培养上清中IL12和γ干扰素(IFNγ)水平,以冻融肝癌细胞株HepG2(1×107个/ml)所获得的肿瘤相关抗原(TAA)刺激DCIL12,MTT法检测TAA负载的DCIL12刺激同源T淋巴细胞(1×105个/ml)增殖分化能力,细胞毒试验检测DCIL12诱导的细胞毒T淋巴细胞(CTL)及其上清液对HepG2肝癌细胞株杀伤作用,ELISA法检测CTL上清液IFNγ水平。结果DC经IL12基因修饰后48h分泌高水平IL12(24.35±5.40)ng/L及IFNγ(725±30)ng/L,均显著高于未转染DC组(P<0.01及P<0.05)。DCIL12诱导的CTL及其上清液对HepG2均有显著杀伤作用,杀伤率显著高于未转染DC组,分别为(82.43±8.70)%vs(57.4±4.3)%(P<0.01)和(76.45±8.50)%vs(18.23±5.30)%(P<0.01),DCIL12诱导的CTL上清液IFNγ水平显著高于未转染DC组,分别为(1125.0±32.7)ng/Lvs(281.3±14.7)ng/L(P<0.01)。结论IL12基因修饰增强DC体外诱导自体T淋巴细胞产生特异性抗肝癌免疫,其机制与IL12基因修饰促进DC分泌IL12、增强T淋巴细胞活化及分泌IFNγ密切相关。
Objective To investigate the efficacy and mechanism of retrovirus transduction of interleukin (IL) 12-transfected dendritic cells (DCs) in vitro. Methods The infection index (MOI) was 100, DCIL12 was modified with recombinant retrovirus containing IL12 and DC12 in culture supernatants of DCIL12 (5 × 103 cells / ml) was detected by enzyme linked immunosorbent assay (ELISA) (TAA) obtained from freezing-thawing hepatoma cell line HepG2 (1 × 107 cells / ml) stimulated DCIL12 and TAA-loaded DCIL12 stimulated homologous T lymphocytes 1 × 105 cells / ml). Cytotoxicity assay was used to detect the killing effect of DCIL12-induced cytotoxic T lymphocytes (CTLs) and their supernatants on HepG2 hepatoma cell lines. ELISA method was used to detect IFNγ levels in CTL supernatants. Results High levels of IL12 (24.35 ± 5.40) ng / L and IFNγ (725 ± 30) ng / L secreted by DC12 at 48h were significantly higher than those of untransfected DCs (P <0.01 and P <0.05). DCIL12 induced CTL and its supernatant had a significant killing effect on HepG2, the killing rate was significantly higher (82.43 ± 8.70)% vs (57.4 ± 4.3)% (P <0.01) and ( The levels of IFNγ in CTL supernatants induced by DCIL12 were significantly higher than those in non-transfected DCs (1125.0 ± 32.7 ng / L vs 281.3 ± 14.7% vs 76.45 ± 8.50% vs 18.23 ± 5.30%, P <0.01) ) ng / L (P <0.01). CONCLUSION: The IL12 gene modification enhances the specific anti-hepatoma immunity induced by DC in vitro induced by DC. The mechanism of IL12 gene modification is closely related to IL12 gene modification, which can promote the secretion of IL12 by DCs, enhance the activation of T lymphocytes and secrete IFNγ.