目的：研究携带hTNF-α基因的第二代重组腺病毒对人肺腺癌细胞系Ani973的体外生物学特性的影响，探讨重组腺病毒在体内抗肿瘤作用。方法：将携带有hTNF-α基因的重组腺病毒（rAd-hTNF-α）感染人肺腺癌细胞系，通过细胞生长实验、克隆形成实验、流式细胞分析、形态学检查等观察其对Anip973肿瘤细胞的作用；利用PCR技术对转染后的细胞进行检测并应用ELISA试剂盒检测转染后的细胞TNF-α的分泌量；通过肿瘤局部注射rAd-LacZ,rAd-hTNF-α的方法观察其在小鼠体内的抗肿瘤作用。结果： rAd-hTNF-α经扩增、纯化后，滴度可达10 10PFU/ml，在30 MOI时，对Anip973细胞的转染率达90%以上。转染的Anip973肿瘤细胞除表面结构和超微结构有轻微变化外，其生长能力、克隆形成率及细胞周期等无明显变化。24 h细胞培养上清TNF-α的分泌量为20 pg/ml细胞。体内实验表明注射rAd-LacZ，rAd-hTNF-α的小鼠肿瘤生长缓慢，体积明显小于对照组（P<0.05），生存期显著延长。结论：腺病毒介导的细胞因子基因hTNF-α转染的Anip973肿瘤细胞未见明显变化，而体内实验则有明显的抗肿瘤作用。

Objective: To study the biological characteristics of tumor cells infected with recombinant adenovirus expressing hTNF-α, investigate the antitumor effect of recombinant adenovirus. Methods: Human lung adenocarcinoma cell line Anip973 was infected with recombinant adenovirus expressing hTNF-α. Cell growth assay, colone formation test, flowcytometry assay and morphology were used to observe the effects on tumor cells. The hTNF-α gene, which was transduced into cancer cells mediated by recombinant adenovirus, was detected by PCR and agarose gel electrophoresis and its products were detected by ELISA assay. The intratumoral injection of rAd-LacZ and rAd-hTNF-α was carried out to evaluate their antitumor effects. Results:The titer of rAd reached 10 10 PFU/ml and more than 90% Anip973 cells could be infected by 30MOI rAd. Except the surface structure and ultrastructure of tumor cells infected with rAd had a light change, cell growth abillity assay, colone formation test, flow cytometry assay showed no significant difference compared with that of the control cells. The TNF-α gene expression at 24 h increased greatly. Antitumor study indicated that on the tumor-bearing mice treated with rAd the tumor grew slowly. Tumor volume was significantly smaller and survive time was prolonged than that of controls.Conclusion:There was no significantly changes occurred on tumoral cells after infected with recombinant adenovirus expressing hTNF-α. The intratumoral injection of rAd-LacZ and rAd-hTNF-α could inhibit the growth of solid tumor.

本课题受黑龙江省自然科学基金资助（D0122）