膜结合配体依赖的TRAIL抗肿瘤细胞旁观者效应

目的：研究TRAIL基因抗肿瘤作用旁观者效应的发生机制。方法：腺病毒Ad/Lac-Z转染肿瘤细胞后作为标记的旁观者细胞，腺病毒Ad/g-TRAIL 转染肿瘤细胞作为效应细胞，在6孔Transwell培养板上同一孔内应用隔离或混合培养旁观者细胞和效应细胞的方法，通过检测旁观者细胞Lac-Z酶的活性，以明确TRAIL基因旁观者效应的发生机制。结果：检测了不同来源的肿瘤细胞株，包括大肠癌细胞株DLD1和Lovo、肺癌细胞株A549、肝癌细胞株HepG2、乳腺癌细胞株MDA-MB231、卵巢癌细胞株DOV13。各株肿瘤细胞转染Ad/g-TRAIL后ELISA方法均未检测出培养液中出现可溶性TRAIL（s-TRAIL）；隔离培养效应细胞与旁观者细胞，旁观者细胞的Lac-Z活性与PBS及腺病毒Ad/CMV-GFP对照组无明显差异，而混合培养组其活性明显低于对照组（P<0.05）；混合培养效应细胞与旁观者细胞（1∶1），发现高密度细胞生长组其旁观者效应明显大于低密度生长组（P<0.05 ）；FACS检测Sub G0/G1 发现DOV13细胞在高密度生长组显著高于低密度生长组（P<0.05）；荧光及相差显微镜下观察发现TRAIL转染细胞可诱导周围旁观者细胞发生凋亡而自己并不首先发生凋亡。结论：腺病毒介导的TRAIL基因的旁观者效应是通过TRIAL蛋白表达并定位于细胞膜上，通过细胞接触结合于邻近肿瘤细胞膜上的TRAIL受体而诱导旁观者肿瘤细胞的凋亡，而不是通过水解膜配体形成s-TRAIL而发生旁观者效应，这种作用是细胞接触依赖的。

AMembrane-bound ligand-dependent bystander effect of the TNF-related apoptosis-inducing ligand (TRAIL)

AIM: To investigate the mechanism of bystander effect caused by TRAIL gene.METHODS: Bystander target cells were transduced with an adenovector expressing the lacZ gene(Ad/Lac-Z),while effector cells were transduced with an adenovector expressing the green fluorescent protein(GFP)/TRAIL fusion gene(Ad/g-TRAIL).Effector and target cells were cocultured in the same well with or without effector and target cell contact using a Transwell plate.RESULTS: A series of different cancer cell lines of different tissue origin were tested for the bystander effect,including human colon cancer cell lines DLD1,human lung cancer cell line A549,human hepatoma cell line Hep G2,human breast cancer cell line MDA-MB231 and human ovarian cancer cell line DOV13.No detectable soluble TRAIL(s-TRAIL) was found from the TRAIL-expressing cell cultures in all of the cell lines.Target cells were killed only when effector and target cells were mixed.The bystander effect and apoptosis induction of TRAIL was dramatically reduced if cells were seeded at a very low density.FACS analysis showed a high apoptosis percentage in high density group in DOV13,morphologically,the effector cells were surrounded by the apoptotic bystander cells.CONCLUSION: The bystander effect of the TRAIL gene is mainly mediated by membrane-bound TRAIL on the surface of transduced cells through cellcell contacts.