免疫基因介入疗法治疗大鼠转移性肝癌的实验研究

Treatment of Metastatic Liver Cancer in Rat by Intraarterial Injection of Cytokine Recombinant Adenoviruses

目的 :观察经动脉插管导入肿瘤坏死因子α(TNF -α)和白介素2(IL -2)重组腺病毒对转移性大鼠肝癌模型的治疗作用。材料和方法 :用293细胞扩增人TNF -α和IL -2重组腺病毒 ,并测定病毒滴度 ;9组大鼠于胃十二指肠动脉分别注入Ad.lacZ(2×109pfu/ml) ,采用X -Gal染色方法检测lacZ基因在各个脏器的表达 ;Walker256乳癌细胞感染人TNF重组腺病毒后 ,观察其体外增殖能力、皮下致瘤性的变化及进行肿瘤局部病理学检查 ;瘤体局部注射TNF重组腺病毒 ,观察荷瘤大鼠的肿瘤生长情况及生存期 ;取大鼠乳腺癌细胞株(Walker256)转染至肝脏的荷瘤大鼠动物模型 ,分为hTNF组 ,人IL -2(hIL -2)组 ,hTNF +hIL -2组 ,病毒对照组(lacZ组)和培养液对照组进行治疗 ,观察大鼠生存期。结果 :所制备的人TNF -α和IL -2重组腺病毒滴度为2×109pfu/ml和2.1×109pfu/ml;实验组于12h取材 ,经染色lacZ基因在肝脏中即有表达 ,而此时脾、肺、肾等内脏器官尚未见表达 ,21d该基因在肝脏仍有表达 ,而在其他脏器如脾、肺及肾表达仅维持14d ;转染基因瘤株体外增殖能力和皮下致瘤性显著下降 ,瘤体周围有大量淋巴细胞及单核细胞浸润 ;肿瘤局部注射Ad.hTNF能显著抑制Walker256的生长 ,并延长荷瘤大鼠的生存期 ;单独注射TNF -α或IL

Purpose:To investigate the therapeutic effects of TNF and IL-2 recombinant adenoviruses via intra-arterial injection on metastatic liver cancer in rat model.Materials and methods:Recombinant adenoviruses harboring hTNF-α or hIL-2 gene were amplified in 293 cells and subjected to titration by the pathogenetic effects on 293 cell.Rats were injected with Ad.LacZ(2×109pfu/ml)through gastra-intestinal artery, liver,spleen,lung and kidney were sampled at 12h,18h,72h,7d,14d,21d and 28d respectively,with X-gal staining was used to check up expression of lacZ gene.Walker 256 cells were infected with human TNF recombinant adenoviruses in vitro,and their proliferation in vitro and tumorigenicity were studied;Walker 256tumor-bearing rats were administered with TNF adenoviruses intratumorally,and the tumor growth and animal survival were monitored.Rats bearing metastatic liver cancer of Walker 256 breast carcinoma were randomly grouped and administered via gastra-intestinal artery with hTNF-α recombinant adenovriuses alone,or hIL-2 recombinant adenovriuses alone,or at the dose of 1.0×109pfu/rat.The therapeutic effects were observed including their survival time.Results:Expression of lacZ gene was detected in liver 12h after injection,but none were done in spleen,lung and kidney.21d lacZ gene expressed in liver,and spleen,lung,kidney and lasted only 14d.After infected with TNF recombinant adenoviruses in vitro,the proliferation capacity of but walker 256 cells decreased in vitro without obvious morphological changes.The tumorigenicity of TNF gene-transfected Walker 256 cells decreased markedly with amounts of lymphocytes and monocytes infiltrating in tumor tissues.Intratumoral injection of TNF recombinant adenoviruses inhibited tumor growth potentially and eventually resulted in tumor regression.The prepared recombinant adenoviruses of hTNF-α and hIL-2 with the titers of 2.0×109pfu/ml and 2.1×109 pfu/ml respectively.1.0×109 pfu hTNF was proper dose.Administration of hTNF-α or hIL-2 recombinant adenoviruses via hepatic artery could extend the survival time of metastatic liver cancer-bearing rats,with the better therapeutic effects achieved by combinational administration of these two adenoviruses.Conclusion:Arterial administration of adenoviruses should be an effective approach in therapy for cancer.