【摘要】 目的构建表达人内皮抑素的重组真核表达载体,研究阳离子脂质体介导转染内皮抑素基因对裸鼠人肝癌生长的抑制作用。方法将含有IL-2信号肽和人内皮抑素基因全长cDNA插入真核表达载体pcDNA3.0产生重组质粒pCD-sEndo,脂质体Dosper介导将pCD-sEndo质粒转染至人肝癌细胞株SMMC-7721,RT-PCR和Western blot检测内皮抑素的表达。建立裸鼠人肝癌模型,按随机数字表法随机分成4组,分别瘤内注射Dosper+pCD-sEndo(Dosper+pCD-sEndo组)、Dosper+pcDNA3.0(Dosper+pcDNA3.0组)、Dosper(Dosper组)和生理盐水(生理盐水组),分时段测量肿瘤的体积;注射结束后1周处死动物,切取肿瘤,免疫组化检测肿瘤组织微血管密度(MVD),TUNEL染色检测肿瘤细胞凋亡指数(AI)。结果成功构建携带人内皮抑素基因和IL-2信号肽的真核表达质粒pCD-sEndo并经酶切鉴定证实;体外转染内皮抑素基因的SMMC-7721细胞,RT-PCR可以检测到内皮抑素mRNA表达,Western blot显示转染细胞培养液中有内皮抑素蛋白表达,而转染空白质粒者中没有;体内实验中,Dosper+pCD-sEndo组裸鼠肿瘤生长受抑,于第12 d起明显小于Dosper+pcDNA3.0组、Dosper组和生理盐水组(P<0.05)。Dosper+pCD-sEndo组平均MVD为6.2±2.5,明显低于生理盐水组(32.8±6.4)、Dosper组(27.8±6.4)和Dosper+pcDNA3.0组(25.5±5.5),P<0.05;Dosper+pCD-sEndo组肿瘤细胞平均AI为24.5±7.3,生理盐水组、Dosper组和Dosper+pcDNA3.0组分别是7.6±2.5、9.5±3.0和11.2±3.6,前者明显高于后三者(P<0.05)。结论瘤内注射携带内皮抑素基因的阳离子脂质体,可减少裸鼠体内种植性人肝癌的微血管数目,促进肿瘤细胞凋亡,抑制肿瘤生长。更多还原

【Abstract】 Objective To construct a recombinant eukaryotic expression vector for human endostatin in order to study the inhibitory effect of liposome-mediated endostatin gene on the growth of human liver carcinoma in nude mice.Methods Human endostatin cDNA including IL-2 secreting peptide was cloned into eukaryotic expression plasmid pcDNA3.0 to construct recombinant plasmid pCD-sEndo.pCD-sEndo plasmid was transferred into hepatocarcinoma cell line SMMC-7721 mediated by liposome Dosper,the expression and secretion of endostatin gene was detected by RT-PCR and Western blot analysis.The suspension of SMMC-7721 cells was injected subcutaneously at the back of 32 nude mice to establish the model of human liver carcinoma.The mice were divided into 4 groups randomly,and injected with Dosper+pCD-sEndo,Dosper+pcDNA3.0,Dosper and physiological brine separately.Tumor volume was measured by stages.The mice were executed after the drug had been given for 1 week,then the microvessel density(MVD) of the tumor tissue was detected with immunohistochemical method and apoptotic index of tumor cells was measured by TUNEL-stain.Results The eukaryotic expression vector pCD-sEndo was successfully constructed and was confirmed by enzyme digestion and sequence analysis.Expression of endostatin gene was detected in transfected SMMS-7721 cells by RT-PCR in vitro,and endostatin protein was also detected in the supernatant of transfected SMMS-7721 cells by Western blot.In vivo study,the growth of human liver carcinoma was inhibited in the group injected with endostatin gene: the average volume of tumor in this group was significantly smaller than that in other groups(P<0.05);the average MVD in this group was 6.2±2.5,significantly less than that in the group injected with physiological brine(32.8±6.4),Dosper(27.8±6.4),or Dosper+pcDNA3.0(25.5±5.5),P<0.05.The average apoptotic index of tumor cells in treatment group,brine group,Dosper group and Desper+pcDNA3.0 group was 24.5±7.3,7.6±2.5,9.5±3.0 and 11.2±3.6 respectively,it was evidently higher in the treatment group than in the latter three groups.Conclusion Human endostatin mediated by cation liposome could decrease the microvessel number of implanted human hepatocarcinoma in nude mice.It could also accelerate apoptosis of tumor cells and inhibit growth of tumor.更多还原