重组腺病毒Ad-CMV-TK的构建及其对Lewis细胞的抑制作用

目的构建重组腺病毒Ad-CMV-TK,并检测Ad-CMV-TK/GCV系统对小鼠Lewis肺癌细胞的抑制作用。方法PCR扩增HSV-TK基因,以复制缺陷型腺病毒为载体构建重组腺病毒Ad-CMV-TK,在HEK293细胞中包装,CsCl2梯度离心法纯化后,感染Lewis肺癌细胞,Western blot检测HSV-TK蛋白的表达;MTT法检测Ad-CMV-TK/GCV系统对Lewis细胞的体外抑制作用;同时检测Ad-CMV-TK/GCV系统对C57荷瘤小鼠的抑瘤作用。结果重组腺病毒Ad-CMV-TK感染的Lewis细胞可特异地表达HSV-TK蛋白,Ad-CMV-TK/GCV系统在体外能明显抑制Lewis细胞的生长,当GCV浓度为100μmol/L时,Lewis细胞的存活率仅为16%;在体内能明显抑制荷瘤小鼠肿瘤的生长,与Ad-Blank/GCV对照组和PBS对照组相比,差异具有统计学意义。结论已成功构建了重组腺病毒Ad-CMV-TK,Ad-CMV-TK/GCV系统在体内及体外对小鼠Lewis肺癌细胞均有明显的抑制作用,为进一步研究其抗肿瘤机制及应用奠定了基础。

Construction of Recombinant Adenovirus Ad-CMV-TK and Its Inhibitory Effect on Lewis Cells

Objective To construct recombinant adenovirus Ad-CMV-TK,and determine the inhibitory effect of Ad-CMV-TK / GCV system on murine lung cancer Lewis cells. Methods Amplify HSV-TK gene by PCR to construct recombinant adenovirus Ad-CMV-TK using replication-defective adenovirus as vector. Ad-CMV-TK was packed in HEK293 cells and purified by cesium chloride gradient centrifugation. Infect Lewis cells with the purified Ad-CMV-TK and determine the expressed HSV-TK protein by Western blot. Ad-CMV-TK / GCV was determined for inhibitory effect on Lewis cells by MTT method,and evaluated for tumorinhibiting effect in tumor-bearing C57 mice. Results HSV-TK protein was expressed in Lewis cells infected with recombinant adenovirus Ad-CMV-TK. Ad-CMV-TK / GCV system significantly inhibited the growth of Lewis cells in vitro. When the concentration of GCV was 100 μmol / L,the survival rate of Lewis cells was only 16%. The tumor sizes of mice treated with Ad-CMV-TK / GCV were significantly smaller than those with Ad-Blank / GCV and PBS controls. Conclusion Recombinant adenovirus Ad-CMV-TK was successfully constructed. Ad-CMV-TK / GCV system inhibited both in vitro and in vivo growths of Lewis cells significantly,which laid a foundation of further study on anti-tumor mechanism of the system.