Wang Xuan, Liu Fukun, Li Xi, Li Jieshou, Xu Genxing
Research Institute of General Surgery, Nanjing General Hospital, Clinical School of Medicine, Nanjing University, Nanjing 210002, China.
Chin Med J (Engl). 2002 Nov;115(11):1664-9.
To explore the inhibitory effect of human endostatin gene mediated by retroviral vector on the growth of human liver carcinoma.
A recombinant retroviral plasmid containing human endostatin gene and signal peptide was engineered and transferred into PA317 cells to produce retrovirus. Human liver carcinoma cells (SMMC7721) were infected with the above retrovirus to build a stable endostatin-transfected liver carcinoma cell line (SMMC-endo). The control liver carcinoma cell line (SMMC-pLncx) was developed in a similar way except that the plasmid was replaced by an empty retroviral vector. Immunohistochemistry and Western blot were used to test the expression and secretion of human endostatin. The biological activity of the expressed human endostatin was assessed by endothelial cell proliferation assay. The growth rates of SMMC-endo and control SMMC-pLncx cells in vivo and in vitro were also observed.
The expression and secretion of human endostatin by endostatin-transfected SMMC-endo cells were confirmed by immunohistochemistry and Western blot. Compared with the control group, concentrated supernatant of SMMC-endo cells remarkably inhibited the proliferation of human umbilical vein endothelial cells by 48%, significantly higher than the inhibition by the control (10.2%; P < 0.01). The endostatin-transfected SMMC-endo cells had similar in vitro growth rates to SMMC-pLncx cells. The in vivo experiment showed that the growth rate of SMMC-endo cells was slowed. Only in 3 out of 5 mice were tumors formed and flank tumors of SMMC-endo cells were 94.5% smaller than those of control cells 22 days after inoculation into nude mice (P < 0.001).
Gene transfer of human endostatin mediated by retroviral vector is an effective form of cancer therapy.
探讨逆转录病毒载体介导的人内皮抑素基因对人肝癌生长的抑制作用。
构建含人内皮抑素基因及信号肽的重组逆转录病毒质粒，并将其转染至PA317细胞以产生逆转录病毒。用人肝癌细胞（SMMC7721）感染上述逆转录病毒，构建稳定转染内皮抑素的肝癌细胞系（SMMC-endo）。以空逆转录病毒载体代替质粒，用类似方法构建对照肝癌细胞系（SMMC-pLncx）。采用免疫组织化学和蛋白质印迹法检测人内皮抑素的表达及分泌情况。通过内皮细胞增殖试验评估所表达的人内皮抑素的生物学活性。同时观察SMMC-endo细胞和对照SMMC-pLncx细胞在体内和体外的生长速率。
免疫组织化学和蛋白质印迹法证实了转染内皮抑素的SMMC-endo细胞可表达及分泌人内皮抑素。与对照组相比，SMMC-endo细胞的浓缩上清液可显著抑制人脐静脉内皮细胞的增殖，抑制率达48%，明显高于对照组（10.2%；P<0.01）。转染内皮抑素的SMMC-endo细胞在体外的生长速率与SMMC-pLncx细胞相似。体内实验显示，SMMC-endo细胞的生长速率减慢。接种至裸鼠体内22天后，5只小鼠中只有3只形成肿瘤，且SMMC-endo细胞的胁腹肿瘤比对照细胞小94.5%（P<0.001）。
逆转录病毒载体介导的人内皮抑素基因转移是一种有效的癌症治疗方式。
