Availability of interlrulkin 12 for gene therapy of hepatoma

作为与DR的联合研究，我们研究了白介素12用于肝癌基因治疗的有效性。艾达·T·布鲁姆在美国FDA工作。用硫代乙酰胺复制大鼠肝硬变模型，观察IL-12对人肝癌的治疗作用。我们将AH66F细胞，即同基因肝癌细胞，植入大鼠肝被膜下，并进行了IL-12的腹腔注射。给药后7周测得肿瘤重量为0.7g，显著低于对照组(2.2g)。相反，IL-12在体内给药对肝脏有不良影响。为了减少IL-12的剂量，我们研究了IL-12和IL-18的协同作用，IL-12和IL-18可以诱导NK或T细胞上的IL-12受体。IL-18增强IL-12介导的人NK细胞杀伤活性。我们研究了NK细胞具有高细胞毒活性的细胞内最佳微环境。NK-CEL-…的毒性研究肝硬变大鼠肝和人肝切除后，肝硬变和肝癌的细胞明显受损，谷胱甘肽前体N-乙酰半胱氨酸(NAC)使其体外活性恢复到正常水平。此外，体内应用IL-12和NAC均可抑制肝硬变大鼠肝脏移植瘤的进展(0.3g)。另一方面，我们计划将IL-12基因导入肝细胞。IL-12基因(p40和p35由美国遗传研究所提供。作为初步检测，制备了带有鸡β-肌动蛋白启动子的β-半乳糖苷酶基因(LacZ)和FITC标记的寡核苷酸(FITC-ODN)。分别将含有磷脂酰丝氨酸或DC胆固醇的干脂混合制成脂质体或阳离子脂质体。将这些悬液与DNA或FITC-ODN孵育，并灭活HVJ。将这些脂质体复合物分别导入人肝癌细胞株Hep3B和HuH7细胞。将其直接注射入Wistar大鼠体内肝脏内。为了检测细胞对FITC-ODN的摄取，在荧光显微镜下观察细胞或肝脏的摄取情况。注射含LacZ的HVJ-脂质体72小时后进行X-Gal染色。用含LacZ的HVJ-脂质体转染人肝癌细胞株Hep3B和Huh7后，仅有2%或3%的细胞X-Gal染色阳性。而含EITC-ODN的HVJ-阳离子脂质体转染后30min，几乎所有细胞都能检测到荧光，尤其是50-60%的细胞核聚集。含LacZ的HVJ-脂质体转染组大鼠肝细胞X-Gal染色阳性细胞数为57%。相反，含有FITC-ODN的HVJ-阳离子脂质体在体内仅在非实质细胞中观察到荧光，可能是由于非实质细胞的干扰。虽然免疫印迹法和ELISA法均未检测到IL-12的表达，但用脂质体的方法将IL-12与IL-18或NAC联合治疗，可能会介导肝淋巴细胞的最佳细胞毒活性。较少

We investigated the availability of ilterleulkin (IL) -12 for the gene therapy of hepatoma as a joint research with Dr,. Eda T. Bloom in FDA in the United State. We made a rat cirrhotic liver with use of thioacetamide as a in vivo model to evaluate the efficacy of IL-12 for the therapy of human hepatoma. We implanted AH66F cells, syngenic hepatoma cells, beneath the capsule of rat liver and performed the intra-peritoneal administration of IL-12. Tumor weight measured 7 weeks after administration was 0.7g, which was significantly lower than control (2.2g). On the contrary, IL-12 has an adverse effect to the liver when administrated in vivo. To reduce the dose of IL-12, we examine the synergistic effect of IL-12 and IL-18, which can induce IL-12 receptors on NK or T cells. IL-18 enhanced IL-12 mediated cytotoxic activity of separated human NK cells. We investigated the intracellular optimal microenvironment where NK cells can have high cytotoxic activity. The citotoxic activity of NK cel … More ls from cirrhotic rat liver or resected human liver bearing cirrhosis and hepatoma was significantly impaired, N-acetylcystein (NAC), the precursor of glutathione, resored their activity up to normal level in vitro. Further more, in vivo administration of both IL-12 and NAC suppressed the progression of tumors implanted in cirrhotic rat liver (0.3g). On the other hand, we planned to introduce IL-12 gene to liver cells. IL-12 gene (p40 and p35 were provided by Genetic Institute, USA. As preliminary assay, β-galactosidase gene with chicken β-actin promoter (LacZ) and FITC labeled oligodeoxynucleotide (FITC-ODN) were prepared. HVJ-liposome or HVJ-cationic liposome was prepared as follows ; dried lipids containing phosphatidylserine or DC cholesterol were mixed for liposome or cationic-liposome, respectively. These suspensions were incubated with DNA or FITC-ODN and inactivated HVJ. Hep3B and Huh7 (human hepatoma cell line) were transfected with these liposome complexes. These were directly injected into Wister rat liver in vivo. In order to detect the cellular uptake of FITC-ODN, transfected cells or livers were examined by fluorescent microscopy. X-gal staining was performed 72 hours after the injection of HVJ-liposome containing LacZ. After transfection of HVJ-liposome containing LacZ, only 2 or 3% of the Hep3B and Huh7 cells were positive in X-gal staining. However, fluorescence was detected in almost all cells even at 30 minutes after transfection of HVJ-cationic liposome containing EITC-ODN, especially 50-60% of nucleus of cells were accumulated. X\gal staining of rat liver transfected with HVJ-liposome containing LacZ revealed that 57% cells of hepatocytes were positive. In contrast, fluorescence was observed only in non-parenchymal cells in vivo after transfection of HVJ-cationic liposome containing FITC-ODN, probably due to interference by non-parenchymal cells. Although IL-12 expression was not detected by Western blotting nor ELLISA by now, the combination therapy of IL-12 by HVJ-liposome method with IL-18 or NAC may mediate the optimal cytotoxic activity of liver lymphocytes. Less