肿瘤选择性增殖腺病毒CNHK300治疗乳腺癌的体外实验研究(英文)

目的观察肿瘤选择性增殖腺病毒 CNHK300对乳腺癌的选择性杀伤作用。方法用 RT-PCR 方法检测各种细胞株的端粒酶活性;CNHK300、ONYX-015(E1B 55 KDa 蛋白缺失的2型和5型嵌合型腺病毒)、wtAd5(野生型腺病毒)分别行病毒增殖实验和细胞生长抑制实验,验证 CNHK300选择性复制和杀伤能力;Western Blot 检测腺病毒E1A 在细胞中的表达。结果乳腺癌细胞株 MCF-7、BT-549和 SK-BR-3端粒酶 hTERT mRNA 均为阳性表达,而正常成纤维细胞株 MRC-5和 BJ 端粒酶hTERT mRNA 为阴性。CNHK300在乳腺癌细胞 MCF-7、BT-549和 SK-BR-3中48 h 复制倍数分别为40 625、1265和20000倍,与wtAd5的增殖能力相似,较 ONYX-015增殖能力强,在 MCF-7和 BT-549细胞中复制能力甚至强于野生型腺病毒。然而,在正常成纤维细胞 MRC-5和 BJ 中 CNHK300病毒增殖能力减弱,48 h 增殖倍数为63～192倍,而 wtAd5增殖仍可高达3160～4846倍CNHK300 MOI 10 PFU/cell 作用7天,可有效杀伤半数乳腺癌细胞,与 ONYX-015相比,CNHK300具有更强的肿瘤杀伤能力CNHK300对正常成纤维细胞的杀伤力较 wtAd5明显减弱,CNHK300在 MOI 100 PFU/cell 时 BJ 细胞存活率50%以上。正常成纤维细胞株中未检测到 CNHK300 E1A 基因表达,在293细胞和感染 CNHK300的乳腺癌细胞株中能够检测到 E1A 基因表达。结论 hTERT 启动子可成功地调控腺病毒 CNHK300选择性在端粒酶阳性的乳腺癌细胞中复制,并产生溶瘤作用。可望成为治疗乳癌的一种新的治疗策略。

Replication-selective Oncolytic Adenovirus CNHK300 in the Treatment of Breast Cancer Cell Lines in vitro

Objective:To evaluate the tumor selectivity and therapeutic efficiency of replication-competent adenovirus CNHK300 on human breast cancer cells.Methods:RT-PCR was used to detect the hTERT mRNA activity in various breast cancer and normal fibroblast cell lines.Virus proliferation assay,cell viability assay and Western blot were applied to evaluate the proliferation and cytolysis selectivity of CNHK300.Results:The telomerase activity of MCF-7,BT-549 and SK-BR-3 was positive,while telom- erase in MRC-5 and BJ was negative.The progeny virus titers in MCF-7,BT-549 and SK-BR-3 after 48 h of CNHK300 exposure was 40 625,1265 and 20000 fold higher than those of 0 h,even slightly higher than those of wtAd5(except in SK-BR-3).ONYX-015 virus proliferation ability was weaker than that of CNHK300 in cancer cells.However,CNHK300 exhibited attenuated replicative ability as compared with wtAd5 in MRC-5 and BJ.The CNHK300 replicatative multiple was 63 and 192 fold at 48 h respectively. while the wtAd5 still multiplied 3 160-4 846 fold.CNHK300 could cause about half of breast cancer cells to die within 7 days at MOI 10 pfu/cell and below,whereas the IC_(50) in BJ and MRC-5 was as high as MOI 100 pfu/cell.CNHK300 E1A protein could be detected in breast cancer cells and 293 cells but not in normal fibroblast cells.Conclusion:hTERT promoter can successfully modulate the CNHK300 to be selectively replicated in breast cancer ceils positive for telornerase,which may be a potential treatment strategy in breast cancer.