TRAIL真核表达质粒的构建及抑制肝癌生长的实验研究

目的构建TNF相关凋亡诱导配体(TRAIL)基因的真核表达质粒，观察其对BALB／c裸鼠肝癌细胞皮下移植瘤模型的抑瘤作用。方法提取U937细胞总RNA．用RT-PCR方法扩增出胞外区(114—281aa)，连入溶菌酶信号肽序列，构建分泌型TRAIL重组质粒；建立裸鼠7402肝癌细胞皮下移植瘤模型，纯化后的质粒DNA与脂质体聚乙烯胺混合后经肌肉注射进行基因转染，体内验证重组蛋白的抑瘤活性，采用TUNEL法进行肿瘤组织细胞凋亡检测。结果肿瘤体积测定结果显示．TRAIL对肝癌细胞生长有抑制作用；凋亡检测光镜下可见有棕褐色凋亡细胞，细胞凋亡指数增高。结论重组TRAIL质粒能引起肿瘤细胞的凋亡，抑制7402肝癌细胞的生长。

Construction of Secreting Plasmid for TRAIL and Its Inhibitory Effect on Liver Cancer

Objective To construct the eukaryotic expressing plasmid of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL),and study its inhibitory effect on hepatic tumor which implanted subcutaneously in nude BALB/c mice.Methods Total RNA of U937 cell was extracted, and its extracellular domain (114-281aa) was amplified by RT-PCR, then signal peptide was ligated. The recombinant secreting plasmid for TRAIL was constructed successfully which was confirmed by enzyme cleavage identification and sequencing identification. Liver cancer cell (strain No.7402) was implanted subcutaneously in 32 nude BALB/c mice. These mice were randomly divided into two groups: study group and control group. The mice in study group received muscular injection of plasmids for transfection, and the mice in control group received the injection of normal saline at the same time. The size of implanted tumors were measured continuously till the day of sacrificing, tumor cell apoptosis effect was examined by TUNEL method. Results In study group,tumor volume was smaller than that in control group and the blue-purple apoptosis cells were observed under microscope. Conclusion TRAIL plasmid can induce apoptosis of liver cancer cell and can inhibit the growth of liver tumor.