增殖型腺病毒AdTPHrehEndo治疗胰腺癌的体外实验研究.docVIP

增殖型腺病毒AdTPHre-hEndo治疗胰腺癌的体外实验研究 　　作者：方益锋，张启瑜，吕和平，单云峰，周蒙滔 作者单位：温州医学院第一附属医院 肝胆外科，浙江 温州 325000 　　【摘要】 目的 研究携带人内皮抑素基因的新型双重调控增殖型腺病毒AdTPHre-hEndo对胰腺癌细胞的体外治疗作用。方法 采用四氮唑盐比色法(MTT)测定不同滴度的病毒AdTPHre-hEndo、Ad-hEndo、ONYX-015、Wad5对胰腺癌细胞和正常细胞的杀伤效应，通过增殖实验观察重组病毒的选择性增殖能力，利用Western blotting和ELISA检测人内皮抑素治疗基因的表达。结果 AdTPHre-hEndo、Ad-hEndo、ONYX-015、Wad5感染胰腺癌细胞杀伤的半数有效量(ED50)分别是MOI=0.624、MOI100、MOI=0.781和MOI=0.284;感染正常细胞时Wad5的ED50为MOI=0.976，余三种病毒均为MOI100。增殖实验结果证实AdTPHre-hEndo可以选择性地在胰腺癌细胞中增殖。随着AdTPHre-hEndo感染时间的延长，肿瘤细胞培养上清液中内皮抑素表达量不断增加，第7天达(310.25±1.41)ng/mL，明显高于感染正常细胞时的表达量(95.24±2.26)ng/mL(P0.01)。结论 AdTPHre-hEndo介导的治疗基因表达量在胰腺癌细胞中可得到放大，在胰腺癌细胞中具有特异性增殖及杀伤能力。 　　【关键词】 腺病毒;内皮抑素;胰腺肿瘤;增殖;基因表达调控，病毒 　　An study for the effect of newly constructed replication-selective adenovirus AdTPHre-hEndo on pancreatic cancer in vitro FANG Yifeng， ZHANG Qiyu， LV Heping， et al. Department of Hepatobiliary Surgery， the First Affiliated Hospital of Wenzhou Medical College， Wenzhou 325000Abstract Objective To investigate the effect of a newly replicating adenovirus vector AdTPHre-hEndo carrying anti-tumor endostatin gene on pancreatic cancer cells. Methods The cytotoxicity of AdTPHre-hEndo on SW1990 and CCC-HPE-2 cells was assessed with a colorimetric assay method and was then compared with that of Ad-hEndo， ONYX-015 and Wad5 on the same cells. The transgene expression of endostatin was detected with Western blotting assay and was also quantitatively analyzed with ELISA assay. Results The cytotoxicity ED50 (50% median effective dose) of AdTPHre-hEndo to normal cell lines CCC-HPE-2 was 160 fold of that to SW1990 cell lines. The tumor-selective cytotoxicity of AdTPHre-hEndo in pancreatic cancer cells was observed. Compared with Wad5， the cytotoxicity of AdTPHre-hEndo was both attenuated in pancreatic cancer cells and normal cells. The cytotoxicity of AdTPHre-hEndo was superior to that of ONYX-015. Proliferative test revealed that AdTPHre-hEndo could proliferate selectively in tumors. Furthermore， in co