Availability of interlrulkin 12 for gene therapy of hepatoma

我们与Dr，共同研究了IL-12在肝癌基因治疗中的应用。埃达·T Bloom在美国的FDA。我们用硫代乙酰胺制作大鼠肝硬化模型，以评价IL-12对人肝癌的治疗效果。我们将AH 66 F细胞（同系肝癌细胞）植入大鼠肝被膜下，并进行IL-12的腹腔内给药。给药后7周测量的肿瘤重量为0.7g，显著低于对照组（2.2g）。相反，IL-12在体内给药时对肝脏具有不利影响。为了减少IL-12的剂量，我们检查了IL-12和IL-18的协同作用，其可以诱导NK或T细胞上的IL-12受体。IL-18增强IL-12介导的分离的人NK细胞的细胞毒活性。我们研究了NK细胞可以具有高细胞毒活性的细胞内最佳微环境。NK细胞的细胞毒活性 ...更多信息 在离体条件下，谷胱甘肽前体N-乙酰半胱氨酸（NAC）的活性恢复到正常水平。此外，在体内施用IL-12和NAC两者抑制了移植在肝硬化大鼠肝脏（0.3g）中的肿瘤的进展。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。作为初步试验，制备了带有鸡β-actin启动子的β-半乳糖苷酶基因（LacZ）和FITC标记的寡核苷酸（FITC-ODN）。如下制备HVJ-脂质体或HVJ-阳离子脂质体;将含有磷脂酰丝氨酸或DC胆固醇的干燥脂质分别混合用于脂质体或阳离子脂质体。将这些悬浮液与DNA或FITC-ODN和灭活的HVJ一起孵育。用这些脂质体复合物转染Hep 3B和Huh 7（人肝癌细胞系）。将这些直接注射到体内Wister大鼠肝脏中。为了检测细胞对FITC-ODN的摄取，通过荧光显微镜检查转染的细胞或肝脏。在注射含有LacZ的HVJ-脂质体后72小时进行X-gal染色。转染含LacZ的HVJ脂质体后，只有2%或3%的Hep 3B和Huh 7细胞在X-gal染色中呈阳性。而含EITC-ODN的HVJ-阳离子脂质体转染30 min后，几乎所有细胞都能检测到荧光，尤其是50-60%的细胞核聚集。用含LacZ的HVJ-脂质体转染大鼠肝细胞，经Xgal染色，阳性率为57%。与此相反，在体内转染HVJ-阳离子脂质体后，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。虽然目前尚未用Western blotting和ELISA检测到IL-12的表达，但HVJ-脂质体法IL-12与IL-18或NAC的联合治疗可能介导肝淋巴细胞的最佳细胞毒活性。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