脾内转染人IL-2与鼠IL-12融合基因对大鼠诱发肝癌的治疗作用

Transfection of interleukin 12 and interleukin 2 fusion gene into spleen for treatment of chemically induced hepatocellular carcinoma in rat

目的研究脾内注射携带人白细胞介素2(hIL-2)与鼠白细胞介素12(mIL1-2)融合基因的逆转录病毒包装细胞株对大鼠诱发肝细胞癌的生长抑制作用。方法构建携带hIL-2和mIL-12融合基因的逆转录病毒载体GCIL1-2EIL-2PN,转染包装细胞PA317后,分别在大鼠肝癌诱发后90天(早期治疗组)及105天(晚期治疗组)进行脾内注射治疗,观察大鼠生存时间及毒性反应。ELISA法检测大鼠血清IL-12和IL-2浓度,放射性活度测定法检测脾脏NK细胞活性。结果IL-12+IL-2联合基因治疗组中,早期治疗及晚期治疗的生存时间分别为188.1±14.2天及168.5±13.6天;IL-12基因治疗组分别为168.2±13.4天及149.1±13.8天(与联合基因治疗组相比,P<0.01);IL-2基因治疗组分别为145.9±11.3天及131.1±10.5天(P<0.01)。3个治疗组中,早期治疗的平均生存时间均长于晚期治疗(P<0.01)。IL-12+IL-2联合基因治疗组中,早期治疗后长期存活率(≥240天)为25%(5/20),治疗后5天肝癌组织中浸润的淋巴细胞明显增多,治疗后2个月血清hIL-2及mIL-12仍维持在较高水平。各基因治疗组NK细胞活性较对照组显著增高(P<0.01)。IL-12+IL-2联合基因组NK细胞活性明显高于单基因治疗组(P<0.01)。结论脾内直接注射携带hIL-2和(或)mIL-12基因的逆转录包装细胞株可显著提高NK细胞的活性,抑制?

Objective To study the inhibitory effect of intrasplenic injection of retroviral packaging cells encoding human interleukin-2 (hIL-2) and mouse interleukin-12 (mIL-12) fusion gene on the growth of chemically induced hepatocellular carcinoma in rats. Methods The retroviral vector GCIL12EIL2PN encoding hIL-2 and mIL-12 fusion gene was constructed. The retroviral packaging cell line PA317 transfected with the vector was injected into the spleens of rats with established chemically induced hepatoma on on the 90th day (early-stage treatment) or the 105th day (late-stage treatment). The survival time and toxic effect were observed. The serum mIL-12 and hIL-2 levels were assayed with ELISA, and the cytotoxicity of the natural killer (NK) cells was measured by means of a 51Cr-release assay using YAC-1 tumor cells as the target. Results The average survival time (after chemical induction) in the early-stage treatment rats and the late-stage treatment rats were 188.1±14.2 days and 168.5±13.6 days, respectively, in IL-12+IL-2 combination gene treatment group, and it was longer than that of IL-12 gene treatment group (168.2±13.4 days and 149.1±13.8 days, respectively, P<0.01), and also IL-2 gene treatment group (145.9±11.3 days and 131.1±10.5 days respectively, P<0.01). The average survival time of the early-stage treatment rats was longer than that of that of the late-stage treatment rats in all gene treatment group (P<0.01). Long survival (≥240 days) rate of the early-stage treatment rats in IL-12+IL-2 combination gene treatment group accounted for 25% (5/20). The pathological study showed that the number of infiltrating lymphocytes in liver tumor tissues was increased obviously 5 days after treatment. The serum hIL-2 and mIL-12 levels in these rats remained high on the 60th day after treatment. Compared with control groups, NK cell activity was increased markedly in every gene treatment groups (P<0.01). NK cells in IL-12+IL-2 gene treatment group were activated significantly compared with IL-12 or IL-2 gene treatment group (P<0.01). Conclusion Both hIL-2 and mIL-12 gene injected intrasplenically can increase the NK cell activity significantly and inhibit the growth of chemically induced hepatocellular carcinoma in rats. The therapeutic efficacy of early administration was superior to that of late treatment, and IL-12+IL-2 fusion gene treatment is more efficacious in activating NK cells than IL-2 or IL-2 gene alone.