Yang Jia-he, Fan Rui-fang, Qian Qi-jun, You Tian-geng, Xue Hui-bin, Su Chang-qing, Cao Hui-fang, Wu Meng-chao
Eastern Hepatobilliary Surgery Hospital, Second Military Medical University Shanghai 200438, China.
Zhonghua Yi Xue Za Zhi. 2003 May 10;83(9):740-3.
To study the inhibitory effect of retroviral packaging cells injected intrasplenically encoding mouse interleukin-12 (mIL-12) and human interleukin-2 (hIL-2) fusion gene on the growth of hepatocellular carcinoma.
The retroviral vectors encoding mIL-12 gene, hIL-2 gene, and mIL-12 and hIL-2 genes, GCIL12EXPN, GCXEIL2PN, and GCIL12EIL2PN were constructed and then transfected into the retroviral packaging cells PA317 to construct cells PA317-GCIL12EXPN, PA317-GCXEIL2PN, and PA317-GCIL12EIL2PN. Rat hepatocellular carcinoma cells CBRH3 were implanted into the livers of Wistar rats to establish hepatoma animal model. Then the rats were divided into 5 groups to be injected intrasplenically with normal saline one day after the implantation (0.8 ml/rat, group I, n = 10), blank vector PA317-GCXEXPN one day after the implantation (10(7) cells/rat, group II, n = 10), PA317-GCIL12EXPN containing IL-2 gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group III, n = 40), PA317-GCXEIL2PN containing mIL-12 gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group IV, n = 40), and PA317-GCIL12EIL2PN containing IL-12-IL-2 fusion gene (1 x 10(7) cells/rat 1, 3, 5, or 7 days after the implantation, group V, n = 40) respectively. The rats surviving longer than 2 months were re-injected with hepatocellular carcinoma cells. The therapeutic effect, immune function and toxic effect were evaluated. CT was conducted on the liver before and after the experiment. Laparotomy was performed 3 and 7 days after treatment to resect some of the carcinoma tissues to undergo pathological examination and OX8 immunohistostaining. Serum mIL-12 and hIL-2 were detected one day before and 3, 7, 30, and 60 days after treatment.
The average survival times of the rats treated with IL-12-IL-2 fusion gene at the first, third, fifth and seventh day after tumor implantation were 53.3 +/- 3.7 days, 49.3 +/- 4.2 days, 31.0 +/- 2.1 days, and 24.3 +/- 1.4 days respectively, longer than those treated with IL-2 gene (25.0 +/- 2.5 days, 23.5 +/- 2.0 days, 18.3 +/- 2.4 days, and 12.0 +/- 1.8 days respectively, P < 0.001), and those treated with IL-12 gene (39.0 +/- 4.8 days, 32.0 +/- 3.9 days, 23.0 +/- 2.5 days, and 19.4 +/- 2.1 days respectively, P < 0.001). Long survival (>or= 60 days) rate in the rats treated with IL-12-IL-2 fusion gene on the first and third day was 30%. The serum mIL-12 and hIL-2 levels in these rats remained high on the 60th day after treatment. The pathological study showed that the number of infiltrating lymphocytes in liver tumor tissues was increased in the IL-12-IL-2 fusion gene treatment group.
The retroviral packaging cell line injected intrasplenically encoding mIL-12 and hIL-2 fusion gene inhibits the growth of hepatocellular carcinoma significantly in rats. The therapeutical efficacy of early administration is superior to that of late one.
研究经脾内注射编码小鼠白细胞介素-12（mIL-12）和人白细胞介素-2（hIL-2）融合基因的逆转录病毒包装细胞对肝癌生长的抑制作用。
构建编码mIL-12基因、hIL-2基因以及mIL-12和hIL-2基因的逆转录病毒载体GCIL12EXPN、GCXEIL2PN和GCIL12EIL2PN，然后转染至逆转录病毒包装细胞PA317，构建细胞PA317-GCIL12EXPN、PA317-GCXEIL2PN和PA317-GCIL12EIL2PN。将大鼠肝癌细胞CBRH3接种于Wistar大鼠肝脏，建立肝癌动物模型。然后将大鼠分为5组，于接种后1天经脾内注射生理盐水（0.8 ml/只，Ⅰ组，n = 10）、接种后1天经脾内注射空白载体PA317-GCXEXPN（10⁷ 个细胞/只，Ⅱ组，n = 10）、接种后1、3、5或7天经脾内注射含IL-2基因的PA317-GCIL12EXPN（1×10⁷ 个细胞/只，Ⅲ组，n = 40）、接种后1、3、5或7天经脾内注射含mIL-12基因的PA317-GCXEIL2PN（1×10⁷ 个细胞/只，Ⅳ组，n = 40）、接种后1、3、5或7天经脾内注射含IL-12-IL-2融合基因的PA317-GCIL12EIL2PN（1×10⁷ 个细胞/只，Ⅴ组，n = 40）。存活超过2个月的大鼠再次接种肝癌细胞。评估治疗效果、免疫功能和毒性作用。实验前后对肝脏进行CT检查。治疗后3天和7天剖腹切除部分癌组织进行病理检查和OX8免疫组化染色。治疗前1天及治疗后3、7、30和60天检测血清mIL-12和hIL-2。
肿瘤接种后第1、3、5和7天用IL-12-IL-2融合基因治疗的大鼠平均生存时间分别为53.3±3.7天、49.3±4.2天、31.0±2.1天和24.3±1.4天，长于用IL-2基因治疗的大鼠（分别为25.0±2.5天、23.5±2.0天、18.3±2.4天和12.0±1.8天，P < 0.001），也长于用IL-12基因治疗的大鼠（分别为39.0±4.8天、32.0±3.9天、23.0±2.5天和19.4±2.1天，P < 0.001）。接种后第1天和第3天用IL-12-IL-2融合基因治疗的大鼠长生存（≥60天）率为30%。这些大鼠治疗后60天时血清mIL-12和hIL-2水平仍较高。病理研究显示，IL-12-IL-2融合基因治疗组肝肿瘤组织中浸润淋巴细胞数量增加。
经脾内注射编码mIL-12和hIL-2融合基因的逆转录病毒包装细胞系可显著抑制大鼠肝癌生长。早期给药的治疗效果优于晚期给药。
