Availability of interlrulkin 12 for gene therapy of hepatoma

作为一项联合研究，我们研究了白细胞介素(IL) -12在肝癌基因治疗中的可用性。Eda T. Bloom在美国FDA工作。我们用硫乙酰胺制备大鼠肝硬化肝作为体内模型，评价IL-12对人肝癌的治疗作用。我们在大鼠肝包膜下植入同种肝癌细胞AH66F细胞，并腹腔注射IL-12。给药后7周肿瘤重量为0.7g，明显低于对照组（2.2g）。相反，IL-12在体内给药时对肝脏有不良影响。为了减少IL-12的剂量，我们研究了IL-12和IL-18的协同作用，它们可以诱导NK细胞或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。我们研究了细胞内NK细胞具有高细胞毒活性的最佳微环境。在体外实验中，肝硬化大鼠肝脏和切除的人肝硬化、肝癌NK细胞的毒性明显降低，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，体内给予IL-12和NAC可抑制肝硬化大鼠肝脏植入肿瘤的进展（0.3g）。另一方面，我们计划将IL-12基因引入肝细胞。IL-12基因（p40和p35）由美国遗传研究所提供。初步制备了鸡β-肌动蛋白启动子β-半乳糖苷酶基因（LacZ）和FITC标记的寡脱氧核苷酸（FITC- odn）。hvj -脂质体或hvj -阳离子脂质体的制备方法如下；分别将含有磷脂酰丝氨酸或DC胆固醇的干燥脂质混合成脂质体或阳离子脂质体。这些悬浮液与DNA或FITC-ODN和灭活的HVJ孵育。用脂质体复合物转染Hep3B和Huh7（人肝癌细胞系）。这些药物直接注射到Wister大鼠体内肝脏。为了检测FITC-ODN的细胞摄取，用荧光显微镜检查转染的细胞或肝脏。注射含LacZ的hvj脂质体72小时后进行X-gal染色。转染含有LacZ的hvj脂质体后，只有2%或3%的Hep3B和Huh7细胞在X-gal染色中呈阳性。然而，即使在转染含有EITC-ODN的hvj阳离子脂质体30分钟后，几乎所有细胞都能检测到荧光，特别是细胞核中有50-60%的细胞聚集。用含有LacZ的hvj脂质体转染大鼠肝脏，X\gal染色显示57%的肝细胞呈阳性。而转染含有FITC-ODN的hvj阳离子脂质体后，体内仅在非实质细胞中观察到荧光，可能是受非实质细胞的干扰。虽然目前还没有通过Western blotting和elisa检测IL-12的表达，但hvj -脂质体法联合IL-18或NAC治疗IL-12可能介导了肝脏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