流体力学方法转染IκBαSR对人肝癌裸鼠原位移植瘤的抑制

The inhibitory effect of hydrodynamics-based transfection of IκBα super repressor gene on the orthotopic transplantation tumor model of human hepatocellular carcinomain in nude mice

目的:使用流体力学方法向人肝癌裸鼠原位移植瘤转染含突变型IκBα的重组逆转录病毒质粒pBABE-puro-IκBα super repressor(pBABE-puro-IκBαSR),观察其对移植瘤生长的作用及其相关机制。方法:使用人肝癌细胞株MHCC97-H、MHCC97-L,建立裸鼠人肝癌原位移植瘤模型,将荷瘤鼠分为MHCC97-H组(对照组)、MHCC97-H+IκBαSR组(转染组)、MHCC97-L组(对照组)和MHCC97-L+IκBαSR组(转染组),1周后,使用流体力学方法通过尾静脉向对照组注射pBABE-puro空载体质粒,向转染组注射pBABE-puro-IκBαSR质粒。每周1次,共3次。观察荷瘤鼠的生存率,检测原位移植瘤块的大小、重量、以及转移瘤数目,使用全自动生化检测仪检测血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)水平。采用免疫组化SP法分析核因子(NF)-κBp65蛋白表达,使用Westernblot检测总蛋白中IκBα蛋白含量。结果:pBABE-puro-IκBαSR质粒显著提高荷瘤小鼠的生存率,转染组肿瘤的体积、重量显著小于对照组,抑瘤率分别为29.3%和33.0%(均P<0.05);转染组荷瘤小鼠ALT和AST显著低于对照组(均P<0.05);NF-κBp65在对照组胞浆中及在细胞核中均呈强阳性表达,在转染组胞浆中呈浅棕色,为弱阳性表达,胞核中极少或没有阳性表达,差异具有统计学意义(P<0.01)。Western blot结果显示,转染组IκBα表达水平高于对照组(P<0.05)。结论:通过流体力学方法向人肝癌原位移植瘤转染突变型IκBαSR质粒,可以抑制移植瘤的生长。其机制可能是IκBαSR抑制了NF-κB的活化。

Objective：To investigate the inhibitory effect on the orthotopic transplantation tumor model of human hepatocellular carcinomain using hydrodynamics-based transfection of recombinant retrovirus vector containg IκBα super repressor gene and its possible mechanism. Methods：The animal model of orthotopic transplantation tumor in nude mice was established with MHCC97-H and MHCC97-L cell lines. Tumor-bearing nude mice were divided into 4 groups：MHCC97-H group （control group）,MHCC97-H＋IκBαSR group （transfection group）, MHCC97-L group （control group） and MHCC97-L＋IκBαSR group （transfection group）. After treatment for 7 clays,plasmid pBABE-puro-IκBαSR and plasmid pBABE-puro in PBS solution were diluted according to the mouse weight. Plasmid pBABE-puro-IκBαSR and plasmid pBABE-puro were respectively transferred into transfection group and control group via the tail vein by hydrodynamic injection. The survival rate of nude mice bearing the tumor was observed. The size,weight and volume of tumor in situ and the number of metastatic tumor in liver were examined, and the inhibitory rates of tumor growth were calculated. ALT and AST in sera were observed. Immunohistochemistry was used to detect the expression of NF-κB in liver cancer tissue. IκBα was determined with western blot. Results：Plasmid pBABE-puro-IκBαSR significantly promoted the survival rate and inhibited the level of ALT and AST in transfection groups than in control groups. In transfection groups,the tumors were significantly smaller and lighter than those in control groups, and the inhibition rates were 29.3% and 33.0% respectively. In cellular nucleus of the transfeetion groups ,the expression levels of NF-κB p65 and IκBα were higher than the control groups. Conclusion：Hydrodynamics-based transfection of plasimd pBABE-puro-IκBαSR gene to the orthotopic implant model of human hepatocellular carcinoma can inhibit the tumor. Its mechanism may be that the transfected IκBαSR gene suppress the activation of NF-κB signaling in cancer cells.