Upegui-Gonzalez L C, Ly A, Sierzega M, Jarocki P, Trojan L, Duc H T, Pan Y, Shevelev A, Henin D, Anthony D, Nowak W, Popiela T, Trojan J
Lab. INSERM, Université Paris VII, Hôpital Bichat, Paris, France.
Hepatogastroenterology. 2001 May-Jun;48(39):660-6.
BACKGROUND/AIMS: To investigate the effect of gene therapy for hepatocellular carcinoma based on inhibition of cellular IGF-I expression, the technique of IGF-I triple helix was investigated in mice developing programmed hepatoma.
mhAT1F1 mouse hepatoma cell line was transfected in vitro with IGF-I triple helix expression vector (pMT-AG-TH) or with IGF-I antisense expression vector (pMT-Anti-IGF-I). 10 x 10(6) transfected cells of either triple helix or antisense type were inoculated intraperitonealy into transgenic ATIIITB6 mice developing genetically programmed hepatoma (mice die between the age of 6 and 7 months). In parallel, human cell cultures established from surgically removed hepatomas were investigated.
mhAT1F1 and human primary cell cultures, transfected with pMT-AG-TH or pMT-Anti-IGF-I vectors resulted in total inhibition of IGF-I demonstrated by immunocytochemical and Northern blot techniques. Transfected cells changed their phenotype and recovered major histocompatibility complex I expression showed by fluorescence-activated cell sorting analysis and Western blot. Moreover, two phenomena were observed in IGF-I "antisense" or "triple helix" transfected cells: 1) the apoptosis, demonstrated by TUNEL technique; 2) the presence of IL-6 simultaneously with disappearance of tumor necrosis factor-alpha and IL-10, investigated by reverse transcriptase-polymerase chain reaction technique. In in vivo experiments, injection of murine transfected cells into mice in terminal-phase prolonged their survival 3-4 months in 100% of cases, as well in "antisense" group (8/8) as in "triple helix" group (10/10).
Injection of hepatoma cells transfected with IGF-I triple helix expression vector, and showing immunogenic and apoptotic characteristics, can constitute an effective cellular therapy against hepatocellular carcinoma.
背景/目的：为研究基于抑制细胞胰岛素样生长因子-I（IGF-I）表达的基因治疗对肝细胞癌的作用，在发生程序性肝癌的小鼠中研究了IGF-I三链体技术。
在体外将IGF-I三链体表达载体（pMT-AG-TH）或IGF-I反义表达载体（pMT-Anti-IGF-I）转染mhAT1F1小鼠肝癌细胞系。将10×10⁶个三链体或反义类型的转染细胞腹腔注射到发生基因程序性肝癌的转基因ATIIITB6小鼠体内（小鼠在6至7个月龄时死亡）。同时，对手术切除的肝癌所建立的人细胞培养物进行研究。
用pMT-AG-TH或pMT-Anti-IGF-I载体转染的mhAT1F1和人原代细胞培养物，通过免疫细胞化学和Northern印迹技术显示IGF-I被完全抑制。转染细胞改变了其表型，并通过荧光激活细胞分选分析和蛋白质印迹显示主要组织相容性复合体I表达得以恢复。此外，在IGF-I“反义”或“三链体”转染细胞中观察到两种现象：1）通过TUNEL技术证实的细胞凋亡；2）通过逆转录聚合酶链反应技术研究发现白细胞介素-6的存在同时伴随着肿瘤坏死因子-α和白细胞介素-10的消失。在体内实验中，将小鼠转染细胞注射到终末期小鼠体内，在100%的情况下，“反义”组（8/8）和“三链体”组（10/10）的小鼠存活期延长了3至4个月。
注射用IGF-I三链体表达载体转染的、具有免疫原性和凋亡特征的肝癌细胞，可构成一种有效的肝细胞癌细胞治疗方法。
