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背景肿瘤患者体内的抗肿瘤免疫受到抑制。增强细胞毒性T淋巴细胞(cytotoxicTlymphocyte,CTL)的活性是提高过继免疫治疗效果,促进肿瘤康复的有效途径之一。目的对转导肿瘤坏死因子(TNF-α)基因的CTL的生物学特性及抗肿瘤活性进行研究,探讨细胞因子基因疗法和过继免疫治疗肿瘤的新途径。设计随机区组实验研究。地点和对象实验在广西医科大学医学实验中心完成。从人肝细胞癌组织中分离淋巴细胞,用BEL7404和IFN-г诱导得到CTL。BALB/c裸小鼠28只,雄性,4～6周龄,体质量18～20g,购自北京医科大学实验动物科学部(SPF级,合格证书医动字第01-3048号)干预用重组反转录病毒载体介导对CTL进行TNF-α基因转导。于每只裸鼠右前肢腋窝皮下注射注射BEL7404,建立荷瘤裸鼠模型,28只裸鼠按体质量分为4组,每组7只。观察转基因CTL对肿瘤原位(右前肢腋窝)及异位(左前肢腋窝皮下)注射治疗效果,分别用生理盐水注射治疗作对照。主要观察指标①检测TNF-α基因转导前后CTL的生长形态,增殖能力,TNF-α分泌量、细胞表型。②体外不同作用时间和效靶比浓度对BEL7404的杀伤效应。③各实验组的肿瘤生成时间,生长速度及肿瘤生成率。结果转基因CTL的生长形态、增殖活性及细胞表型与CTL相似,但具有持续、高效表达TNF-α的能力,72h表达量达410
Background Anti-tumor immunity in tumor patients is inhibited. Enhancing cytotoxic T lymphocyte (CTL) activity is an effective way to improve the effect of adoptive immunotherapy and promote tumor rehabilitation. OBJECTIVE: To study the biological characteristics and anti-tumor activity of CTL transduced with tumor necrosis factor (TNF-α) gene and to explore new ways of cytokine gene therapy and adoptive immunotherapy of tumor. Design Randomized Block Experimental Study. Location and object experiment in Guangxi Medical University Medical Experiment Center completed. Lymphocytes were isolated from human hepatocellular carcinoma tissue and CTLs were induced with BEL7404 and IFN-г. BALB / c nude mice 28 male, 4-6 weeks old, body weight 18 ~ 20g, purchased from Beijing Medical University Laboratory Animal Science (SPF level, Certificate Medical Word No. 01-3048) intervention with recombinant Retroviral vectors mediate TNF-alpha gene transduction of CTLs. BEL7404 was injected subcutaneously into the armpit of the right forelimb of each nude mouse to establish a tumor-bearing nude mouse model. 28 nude mice were divided into 4 groups according to body weight, with 7 in each group. The effect of CTL injection on the tumor in situ (right forelimb armpit) and ectopic (left forelimb armpit) injection were observed and compared with normal saline injection. MAIN OUTCOME MEASURES ① CTL growth morphology, proliferation, TNF-α secretion and cell phenotype were measured before and after TNF-α gene transduction. (2) The killing effect of BEL7404 on different time and concentration of effective target in vitro. ③ The experimental group of tumor formation time, growth rate and tumor formation rate. Results The growth morphology, proliferation activity and cell phenotype of transgenic CTL were similar to those of CTL. However, the expression of TNF-α was up to 410