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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(E1B55KDa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;WesternBlot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hTERTmRNA均为阳性表达,而正常成纤维细胞株MRC-5和BJ端粒酶hTERTmRNA为阴性。CNHK300在乳腺癌细胞MCF-7、BT-549和SK.BR.3中48h复制倍数分别为40625、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,48h增殖倍数为63-192倍,而wtAd5增殖仍可高达3160-4846倍。CNHK300MOI10PFU/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MOI100PFU/cell时BJ细胞存活率50%以上。正常成纤维细胞株中未检测到CNHK300E1A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。
Objective To observe the selective killing effect of tumor selective adenovirus CNHK300 on breast cancer. Methods The telomerase activity of various cell lines was detected by RT-PCR. CNHK300, ONYX-015 (type 2 and type 5 chimeric adenovirus lacking E1B55KDa protein) and wtAd5 (wild-type adenovirus) Experiments and cell growth inhibition experiments to verify CNHK300 selective replication and killing ability; Western Blot detection of adenovirus E1A expression in cells. Results The telomerase hTERT mRNA of breast cancer cell lines MCF-7, BT-549 and SK-BR-3 were all positive, while the hTERT mRNA of normal fibroblast MRC-5 and BJ was negative. The replicative multiples of CNHK300 in human breast cancer cells MCF-7, BT-549 and SK.BR.3 at 48h were 40625, 1265 and 20000 times, respectively. Compared with wtAd5, CNHK300 had stronger proliferative ability than ONYX-015. And BT-549 cell replication even stronger than the wild-type adenovirus. However, in normal fibroblasts MRC-5 and BJ, the proliferation of CNHK300 virus was weakened, with a multiplication of 63-192 fold at 48 h, while proliferation of wtAd5 was still as high as 3160-4846 fold. CNHK300MOI10PFU / cell effect of 7 days, can effectively kill half of breast cancer cells, compared with ONYX-015, CNHK300 has a stronger tumor-killing ability. The cytotoxicity of CNHK300 to normal fibroblasts was significantly weaker than that of wtAd5, while the survival rate of BJ cells of CNHK300 at MOI100PFU / cell was over 50%. No CNHK300E1A gene expression was detected in normal fibroblast cell lines, and E1A gene expression was detected in 293 cells and CNHK300-infected breast cancer cell lines. Conclusion hTERT promoter can successfully regulate adenovirus CNHK300 selectively replicate in telomerase positive breast cancer cells and produce oncolytic effect. Is expected to become a new treatment strategy for the treatment of breast cancer.