重组新城疫病毒Anhinga株与TRAIL蛋白协同杀伤肿瘤细胞

Recombinant Newcastle Disease Virus Anhinga Strain and TRAIL Protein Cooperate to Kill Cancer Cells in Culture

将新城疫病毒(Newscastle disease virus,NDV)Anhinga株的全长基因组cDNA克隆质粒、pTM1-L、pTM1-P、pTM1-NP表达质粒共转染稳定表达T7 RNA聚合酶的BSRT7/5细胞,得到拯救NDV病毒.通过PCR、酶切法、测序证明拯救病毒中存在引入的分子标签.通过血凝实验、蚀斑测定证明成功拯救病毒.并研究了该重组病毒对4种不同人肿瘤细胞的体外杀伤效果.首次证明重组Anhinga株对SMMC-7721细胞、A549细胞、HepG2细胞和SH-SY5Y细胞均有杀伤作用.该重组病毒主要诱导SMMC-7721细胞和A549细胞凋亡,诱导HepG2细胞和SH-SY5Y细胞坏死.TNF家族成员TRAIL蛋白可以显著增强NDV杀伤肿瘤细胞的效果.本实验为进一步研究重组NDV用于肿瘤治疗奠定基础.

The plasmid containing the full-length cDNA from Newcastle disease virus（NDV） Anhinga strain was cotransfected with helper plasmids encoding viral nucleoprotein,phosphoprotein and large polymerase protein into BSR T7 /5 cells stably expressing the T7 RNA polymerase.The recombinant virus was rescued and amplified by inoculation of the supernatant from the transfected cells into the allantoic cavity of specific-pathogen free chicken embryos.By reverse transcriptase-PCR（RT-PCR）,restriction digestion and sequencing confirmation,the success of the rescue process was confirmed by identification of the unique molecular tag of the rescued virus.We also measured virus hemagglutination（HA） titer through the hemagglutination test（HA test）,and determined viral titers by plaque-formation units.Thus,we conclude that the virus was rescued successfully from the cDNA.We evaluated the cancer therapeutic potential of the rescued NDV in tumor cells,SH-SY5Y,A549,SMMC-7721 and HepG2 cells.Our data showed that the recombinant NDV could induce apoptosis in A549 and SMMC-7721 cells,and induce necrosis in SH-SY5Y and HepG-2 cells.The inhibitory effects of the recombinant virus on growth of the tumor cells could be enhanced by treatment with Trail,a TNF family member.