脂质体法转染人肿瘤坏死因子-α基因对裸鼠人肝癌移植瘤的抑制效应及其机制

Inhibitory Effect and Mechanism of Human Tumor Necrosis Factor-α Gene Transfected into Human Hepatoma Transplantation Tumor by Lipofectamine^(TM) 2000 in Nude

背景:外源性肿瘤坏死因子(TNF)-α联合化疗药物对肿瘤的疗效较单独应用更佳,为肿瘤治疗提供了新的方向。目的:对裸鼠人肝癌移植瘤模型行脂质体介导的TNF-α基因瘤内转染,研究肝癌移植瘤的生长抑制情况及其机制。方法:经脂质体介导,以真核表达质粒pSVK3-TNF-α分别转染人肝癌细胞株SMMC-7721和裸鼠皮下SMMC-7721细胞移植瘤。测定SMMC-7721细胞的TNF-α浓度,甲基噻唑基四唑(MTT)法测定细胞杀伤率,流式细胞仪和原位末端标记(TUNEL)法检测细胞周期和凋亡情况。结果:TNF-α转基因治疗裸鼠人肝癌移植瘤结束第5d,移植瘤体积为(75.28±35.35)mm3,显著低于对照组的(326.45±103.64)mm3(P<0.05)。TNF-α基因体外转染SMMC-7721细胞24、48、72h后,基因转染组每106个细胞的TNF-α表达量分别为(1680±187)pg、(1702±205)pg和(1650±164)pg,细胞杀伤率分别为(37.1±2.4)%、(79.4±4.3)%和(84.2±4.6)%。基因转染72h后,SMMC-7721细胞增殖指数为(30.5±3.2)%,显著低于对照组的(46.1±3.9)%(P<0.05);凋亡指数为(10.0±2.1)%,显著高于对照组的(2.7±0.4)%(P<0.01)。结论:脂质体介导的TNF-α基因转染裸鼠人肝癌移植瘤可明显抑制肿瘤生长,其机制可能为影响肿瘤细胞生长周期以及诱导肿瘤细胞凋亡。

Background： Exogenous tumor necrosis factor （TNF）-α combined with chemotherapy against cancer was more effective than either single agent alone, which provides a new direction of tumor therapy. Aims： To transfect TNF-α gene into human hepatoma transplantation tumor model by Lipofectamine^TM 2000 in nude mice, and to study the inhibitory effect and mechanism. Methods： Eukaryotic expression plasmid pSVK3-TNF-α was transfected into human hepatoma cell line SMMC-7721 and subcutaneous nodules of SMMC-7721 cell transplantation tumor by Lipofectamine^TM 2000 in nude mice. Level of TNF-α in SMMC-7721 cells was detected. Methyl thiazolyl tetrazolium （MTT） method was used to detect the death rate. Flow eytometry and TdT-mediated dUTP-biotin nick end labeling （TUNEL） were used to assess the cell cycle and apoptosis, respectively. Results： On the fifth day after TNF-α gene transfected into human hepatoma transplantation tumor, the tumor size was （75.28±35.35） mm^3, significantly lower than that in control group [（326.45±103.64） mm^3] （P〈 0.05）. At 24, 48, 72 hours after TNF-α gene transfected into SMMC-7721 cells, the expression of TNF-α gene in gene transfection group was （1680±187） pg, （1702±205） pg and （1650±164） pg per 106 cells, respectively. The death rate was （37.1±2.4）%, （79.4±4.3）% and （84.2±4.6）%, respectively. At 72 hours after gene transfection, proliferation index of SMMC-7721 cells in gene transfection group was （30.5±3.2）%, significantly lower than that in control group [（46.1±3.9）%] （P〈0.05）. Apoptosis index of gene transfected cells was （10.0±2.1）%, significantly higher than that in control group [（2.7± 0.4）%] （P〈0.01）. Conclusions： The TNF-α gene transfection into human hepatoma transplantation tumor by Lipofectamine^TM 2000 in nude mice can markedly inhibit the tumor growth, and its mechanism may be due to inhibition of tumor cell cycle and induction of cell apoptosis.