携带TRAIL的溶瘤腺病毒治疗三阴性乳腺癌的研究

目的：构建携带肿瘤坏死因子相关凋亡诱导配体（tumor necrosis factor-related apoptosis inducing ligand,TRAIL）基因的溶瘤腺病毒p55-hTERT-HRE-TRAIL,评估该病毒在体外和体内对三阴性乳腺癌的杀伤能力.方法：将质粒p55-hTERT-HRE与pPE3-TRAIL通过Lipofectamine 2000共转染至人胚肾293细胞,获得溶瘤腺病毒p55-hTERT-HRE-TRAIL;采用病毒体外增殖实验观察乳腺癌细胞株MDA-MB-231和人正常乳腺细胞株MCF-10A中的增殖情况;采用四甲基偶氮唑蓝（MTT）比色法比较不同感染复数（multiplicity of infection,MOI）的病毒对两种细胞的抑制效应;采用酶联免疫吸附试验（ELISA）法检测病毒感染细胞后上清液中TRAIL的含量,采用蛋白质印迹（Western blotting）法检测细胞内TRAIL蛋白的表达.建立三阴性乳腺癌的原位成瘤模型及左心室注射模拟转移瘤裸鼠模型,应用“活体内光学成像系统”动态观察肿瘤的生长及转移情况.结果：p55-hTERT-HRE-TRAIL感染乳腺癌细胞株后表现出强大的增殖、复制能力,而在人正常乳腺细胞株内增殖并不明显;很低浓度的p55-hTERT-HRE TRAIL就对乳腺癌细胞产生明显的杀伤效应,而对正常乳腺细胞没有明显的杀伤作用;p55-hTERT-HRE-TRAIL感染乳腺癌细胞后,TRAIL蛋白的表达明显增多,而感染正常乳腺细胞后表达的TRAIL维持在较低的水平.乳腺癌原位成瘤模型中,空白对照组的光子数多于其他各组,肿瘤体积大于其他各组（P＜0.05）.左心室注射模拟转移瘤模型中,对照组活体内光学成像与TRAIL治疗组差异有统计学意义,TRAIL治疗组的生存天数延长（P＜0.05）.结论：成功构建高滴度的溶瘤腺病毒p55-hTERT-HRE-TRAIL,该病毒在乳腺癌细胞中具有特异性增殖并杀伤肿瘤细胞的能力.

Effects of the Oncolytic Adenovirus Carrying TRAIL Gene for Tripple Negative Breast Cancer

Objective：To construct oncolytic adenovirus carrying tumor necrosis factor-related apoptosis inducing ligand （TRAIL） gene,and to evaluate its tumor-specific proliferation and killing capacity for tripple negative breast cancer in vitro and in vivo.Methods：The TRAIL gene was cloned into adenovirus shuttle vector p55-hTERT-HRE to form p55-hTERT-HRE-TRAIL.Viral replication experiments were performed to evaluate the proliferation ability of the virus in breast cancer cell line MDA-MB-231 and the normal breast cell line MCF-10A.Methyl triazole tetrazolium（MTT） assay was performed to determine the inhibitory effect of the virus on the two cell lines.Enzyme linked immunosorbent assay（ELISA） was performed to detect the level of TRAIL in the supernatant after cells were infected with virus,and the intracellular TRAIL protein was detected by Western blotting.The athymic mouse model of breast cancer in situ was established and the metastatic breast cancer was imitated by injection in the left ventricle of the mouse.The optical imaging in vivo system was used to monitor the growth and metastasis of tumors.Results：P55-hTERT-HRE-TRAIL proliferated more efficiently in breast cancer cells than in normal breast cells.P55-hTERT-HRE-TRAIL could kill the breast cancer cells at a very low multiplicity of infection（MOI）,while the killing capacity for the normal breast cells was weak.The level of TRAIL increased obviously at 48 hours postinfection in breast cancer cells,but it maintained low level in normal breast cells.In the athymic mice model of breast cancer in situ,the volume and photon number of the tumors in the control group was significantly larger than those in the TRAIL groups（P＜0.05）.The optical imaging in vivo system showed statistical differences between the control group and the TRAIL groups in the athymic mice model of imitated metastatic breast cancer by infusion in the left ventricle,and the mice of the TRAIL groups survived longer （P＜0.05）.Conclusion：The high-titer oncolytic adenovirus p55-hTERT-HRE-TRAIL is successfully constructed.It can proliferate specifically in the breast cancer cells and kill tumor cells.