TRAIL真核表达治疗肝细胞癌作用的研究

The Therapeutic Effects on Hepatocelluar Carcinoma Caused by Eukaryotic Expression of TRAIL

目的 :构建鼠源TRAIL(mTRAIL)真核表达质粒。研究其体内、外表达对肝癌细胞的诱导凋亡作用 ,抑制肝癌生长作用及与重组人FN多肽真核表达质粒pCH5 10协同抑制肝癌生长作用。方法 :采用RT PCR及重组DNA技术构建mTRAIL真核表达质粒 ;体内、外进行基因转染 ;用流式细胞仪检测肝癌细胞凋亡率 ,用TdT mediateddUTPnickendlabeling (TUNEL)法及免疫组织化学技术检测肝癌细胞凋亡 ;小鼠实体瘤块模型研究基因转染抑制肝癌生长作用。结果 :用RT PCR方法自小鼠脾细胞RNA扩增出TRAIL基因的全长cDNA ,并克隆至真核表达载体pcDNA3 .1中获重组质粒pX1;以pX1转染BHK细胞株后攻击肝癌细胞株H2 2细胞 ,可检测到肝癌细胞凋亡 ;肌肉内注射转染质粒DNApX1,通过诱导肿瘤细胞凋亡抑制肝癌生长 ;pX1与FN多肽真核表达质粒pCH5 10有协同抑制肝癌生长作用。结论 :质粒pX1可在细胞及小鼠体内表达 ;体内、外表达可诱导肝癌细胞凋亡并可通过该机制抑制肝癌生长 ;与FN多肽真核表达质粒pCH5

Objective: To construct an eukaryotic expressing plasmid of mouse TRAIL (mTRAIL), and investigate its apoptosis inducing ability to hepatocelluar carcinoma cells in vitro and in vivo , inhibitory effect on the growth of hepatocelluar carcinoma, and its synergism with pCH510, an eukaryotic expressing plasmid of recombinant human FN polypeptide. Methods: The eukaryotic expressing plasmid of mTRAIL was constructed by RT PCR and DNA recombination techniques. Gene transfection was performed in vitro and in vivo . The apoptosis rate of hepatocelluar carcinoma cells was measured by Flow Cytometry. The apoptosis of hepatocelluar carcinoma cells was also detected by TdT mediated dUTP nick end labeling (TUNEL) and histochemistry techniques. The inhibitory effect of gene transfection on solid tumor was observed in mice. Results: The cDNA of mTRAIL was amplified by RT PCR from the RNA of the mouse spleen cells, and cloned into the eukaroytic expressing vector pcDNA3.1. The recombinant plasmid was designated as pX1. The BHK cells transfected with plasmid pX1 could attack H22 hepatocelluar carcinoma cells and induce them into apoptosis. The transfection of plasmid pX1 through injection into mouse muscles could inhibit the growth of hepatocelluar carcinoma by inducing tumor cells into apoptosis. Plasmid pX1 and pCH510 have a synergistic inhibitory effect on the hepatocelluar carcinoma growth. Conclusion: Plamid pX1 could be expressed in cells and in vivo in mouse. The expression of pX1 in vivo and in vitro could induce hepatocelluar carcinoma cells into apoptosis and inhibit the growth of hepatocelluar carcinoma by this mechanism. Plasmid pX1 and pCH510 have a synergistic inhibitory effect on the hepatocelluar carcinoma growth.