肿瘤坏死因子α基因转导的肿瘤浸润淋巴细胞体外生物学活性及不同转输方式的比较(英文)

Bioactivity of in vitro cultured tumor necrosis factor-alfa transduced tumor-infiltrating lymphocytes and therapeutic effects on human brain glioblastoma infused in different ways

背景:利用肿瘤过继免疫和基因转移技术将表达肿瘤坏死因子α的载体导入运载细胞回输到体内,使肿瘤坏死因子α在肿瘤局部高浓度表达,既可增加肿瘤坏死因子α直接杀伤肿瘤细胞的能力,又能减少对其他组织的毒副作用。目的:探讨肿瘤坏死因子α基因转导的肿瘤浸润淋巴细胞体外生物学活性及不同转输方式对荷瘤裸鼠肿瘤细胞的抑制作用。设计:以实验动物为观察对象的随机对照实验。单位:中国医科大学肿瘤研究所。材料:实验于2000-01/2001-12在中国医科大学肿瘤研究所和中国医科大学实验动物部完成。TJ8510细胞(人脑恶性胶质瘤细胞系细胞):天津医科大学总医院神经病学研究所提供;实验动物:先天性无胸腺BALB/C裸鼠36只。方法:在肿瘤坏死因子α反转录病毒转移系统的建立及运载细胞肿瘤浸润淋巴细胞制备的基础上,利用构建的单克隆病毒细胞株PLC-2和PLJC-5将标记基因NeoR和目的基因肿瘤坏死因子α分别导入到肿瘤浸润淋巴细胞中,然后对其进行细胞增殖、肿瘤坏死因子表达、体外抗瘤活性的检测。将36只裸鼠接种肿瘤后随机分为6组,局部转输对照组、局部转输肿瘤浸润淋巴细胞组、局部转输肿瘤坏死因子肿瘤浸润淋巴细胞组、静脉转输对照组、静脉转输肿瘤浸润淋巴细胞组、静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞组,对荷瘤裸鼠的治疗作用进行观察。主要观察指标:①基因转导前后肿瘤浸润淋巴细胞的增殖情况和肿瘤浸润淋巴细胞的肿瘤坏死因子α的表达。②体外抗瘤活性的测定。③动物实验结果。结果:①肿瘤浸润淋巴细胞、NeoR-肿瘤浸润淋巴细胞及肿瘤坏死因子肿瘤浸润淋巴细胞3组细胞的体外增殖活性差异无显著性意义(P>0.05)。②肿瘤浸润淋巴细胞与NeoR-肿瘤浸润淋巴细胞的肿瘤坏死因子α分泌量差异无显著性意义(P>0.05);而肿瘤坏死因子肿瘤浸润淋巴细胞的肿瘤坏死因子α分泌量与NeoR-肿瘤浸润淋巴细胞、肿瘤浸润淋巴细胞相比差异有显著性意义(P<0.01),而且肿瘤坏死因子肿瘤浸润淋巴细胞在体外培养30d,可持续表达高水平的肿瘤坏死因子α。③肿瘤浸润淋巴细胞与NeoR肿瘤浸润淋巴细胞对TJ8510细胞的杀伤活性差异无显著性意义(P>0.05),而肿瘤坏死因子肿瘤浸润淋巴细胞与前两者相比杀伤活性有明显地提高(P<0.01)。④动物实验结果:转输肿瘤坏死因子肿瘤浸润淋巴细胞40d后,肿瘤局部转输肿瘤坏死因子肿瘤浸润淋巴细胞组肿瘤体积小于局部转输对照组犤(307±42),(2048±278)mm3,P<0.01犦,静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞组肿瘤体积小于静脉转输对照组犤(954±195),(1989±305)mm3,P<0.05犦。结论:肿瘤坏死因子α基因转导的肿瘤浸润淋巴细胞能有效地表达肿瘤坏死因子,其在体外和荷瘤裸鼠体内的抗肿瘤效果明显高于肿瘤浸润淋巴细胞。静脉转输人脑胶质瘤肿瘤浸润淋巴细胞对人脑胶质瘤裸鼠皮下实体瘤生长无明显抑制效应,但静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞却能明显抑制肿瘤的生长,局部转输肿瘤坏死因子肿瘤浸润淋巴细胞较静脉转输肿瘤坏死因子肿瘤浸润淋巴细胞对肿瘤生长的抑制效果更为明显,提示过继免疫基因治疗胶质瘤以局部转输方式为佳。

BACKGROUND： Tumor-adopted immunity and gene transduction tech- nique are used to introduce tumor necrosis factor-α vector into carrier cells, which are then re-infused into the body so that cancer cells can be killed by tumor necrosis factor-α more directly and effectively with fewer side effects on the other tissues due to high local expression. OBJECTIVE： To study the bioactivity of in vitro cultured tumor necrosis factor-α transduced tumor-infiltrating lymphocytes as well as the inhibitory effects on cancer cells of cancer-loaded rats infused in different ways. DESIGN： A randomized controlled study based on experimental animals. SEETING： Cancer Research Institute of China Medical University. MATERIALS： This study was carried out at the Cancer Research Institute and the Experimental Animal Department, China Medical University, between January 2000 and December 2001. TJ8510 cell line （human brain glioblastoma cell line） was provided by the Neurological Research Institute of Tianjin Medical University Affiliated Hospital. The experimental animals were 36 BALB/C nude mice congenitally having no thymus. METHODS： Based on the establishment of tumor necrosis factor-α retroviral transduction system and the preparation of carrier cells tumor-infiltrating lymphocytes, the monoclonal virus cell line PLC-2 and PLIC-5 available were used to introduce marked gene NetR and targeted gene tumor necrosis factor-α into tumor-infiltrating lymphocytes, respectively. Then cell proliferation, tumor necrosis factor expression and in vitro antitumor activity were examined. After cancer cell inoculation, the 36 nude mice were randomly divided into 6 groups： local infusion control group, local tumor-infiltrating lymphocytes infusion group, local tumor necrosis factor-tumor-infiltrating lymphocytes infusion group, venous infusion control group, venous tumor-infiltrating lymphoeytes infusion group and venous tumor necrosis factor-tumor-infiltrating lymphoeytes infusion group, and the therapeutic effects on the cancer-loaded mice were observed. MAIN OUTCOME MEASURES： ① Tumor-infiltrating lymphocytes proliferation and tumor necrosis factor-α expression in tumor-infiltrating lymphocytes before and after gene transduction; ② In vitro anti-tumor activity; ③ Outcome of the animal experiment. RESULTS： ① In vitro proliferation of tumor-infiltrating lymphocytes, NeoR-tumor-infiltrating lymphocytes and tumor necrosis factor-tumor-infiltrating lymphoeytes was not significantly different from each other （P 〉 0.05）. ② Tumor necrosis factor-α secretion of tumor-infiltrating lymphocytes and Neo^R-tumor-infiltrating lymphocytes, though not significantly different （P 〉 0.05）, significantly differ from that of tumor necrosis factor-tumor-infiltrating lymphocytes （P 〈 0.01）; moreover, tumor necrosis factor-tumor-infiltrating lymphocytes were found to express higher tumor necrosis factor-α continuously when in vitro cultured for 30 days. ③ Anti-TJ8510 cancer cell activity did not significantly differ between tumor-infiltrating lymphocytes and Neo^R-tumor-infiltrating lymphocytes （P 〉 0.05）, but obviously increased in tumor necrosis factor-tumor-infiltrating lymphocytes （P 〈 0.01）. ④ Outcome of the animal experiment： 40 days after tumor necrosis factor-tumorinfiltrating lymphocytes infusion, cancer size in local tumor necrosis factortumor-infiltrating lymphocytes infusion group was found smaller than that in local infusion control group [（307±42） and （2 048±278） mm^3, P 〈 0.01], and it was also smaller in venous tumor necrosis factor-tumor-infiltrating lymphocytes infusion group than that in venous control group [（954±195）and （1 989±305） mm^3 , P 〈 0.05]. CONCLUSION： Tumor necrosis factor-α gene transduced tumor-infiltrating lymphocytes could effectively express tumor necrosis factor, exerting higher and in vivo anti-tumor effects than tumor-infiltrating lymphocytes in cancer-loaded nude mice. No obvious inhibitory effects on the growth of subcutaneous solid carcinoma could be observed in nude mice after venous infusion of human brain glioblastoma tumor-infiltrating lymphocytes, but the inhibitory effects became obvious due to venous infusion of tumor necrosis factor-tumor-infiltrating lymphocytes and significant due to local tumor necrosis factor-tumor-infiltrating lymphocytes infusion, indicating that local infusion is the preferable way in the treatment of glioblastoma by immuno-gene therapy.