基于黑猩猩腺病毒6型的新型溶瘤病毒抑瘤研究  

Anti-Tumor Effect of a Novel Oncolytic Virus Based on Chimpanzee Adenovirus Type 6

作　　者：王齐 王一涵 黎鹰 邢嫚 周东明 WANG Qi;WANG Yi-han;LI Ying;XING Man;ZHOU Dong-ming(Department of Pathogenic Biology,School of Basic Medical Sciences,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津医科大学基础医学院病原生物学系,天津300070

出　　处：《四川大学学报（医学版）》2022年第1期71-76,共6页Journal of Sichuan University(Medical Sciences)

摘　　要：目的以6型黑猩猩腺病毒(AdC6)为载体,构建一组新型溶瘤腺病毒,使之获得瘤内特异性复制能力。在体内外检测其抑瘤效果,并探讨其溶瘤机制。方法以AdC6载体为基础,以人端粒酶逆转录酶(hTERT)启动子驱动该腺病毒的复制相关基因E1A表达,获得重组溶瘤病毒AdC6-htertE1A-ΔE3,同源构建表达粒细胞-巨噬细胞集落刺激因子(GM-CSF/CSF_(2))的AdC6-htertE1A-ΔE3(CSF_(2))、复制缺陷型的腺病毒AdC6-ΔE1-ΔE3。重组腺病毒在HEK293细胞中包装,纯化后进行酶切鉴定。以上述3种腺病毒感染不同种类的肿瘤细胞(RD、SW-620、HeLa、Huh7、RM-1与MC-38),24 h后Western blot检测CSF_(2)的表达,72 h后CCK8实验检测肿瘤细胞的存活率。以上述3种腺病毒感染HeLa细胞,12 h、24 h、48 h后以Western blot检测凋亡信号通路蛋白表达水平。在C57BL/6小鼠背部皮下注射含有1×10^(6)个小鼠结肠癌MC38细胞或小鼠前列腺癌RM-1细胞的细胞悬液,建立2种荷瘤小鼠模型实验,分为4组,每组分别瘤内注射50μL的PBS、1×10^(8) PFU的AdC6-ΔE1-ΔE3、AdC6-htertE1A-ΔE3、AdC6-htertE1A-ΔE3(CSF_(2))。当PBS组荷瘤小鼠的肿瘤达到2500 mm^(3)以上时统一处死小鼠,取肿瘤组织进行TUNEL染色,在显微镜下观察凋亡阳性细胞并计数。结果酶切鉴定发现,成功构建了溶瘤病毒AdC6-htertE1A-ΔE3、AdC6-htertE1A-ΔE3(CSF_(2))及AdC6-ΔE1-ΔE3。Western blot检测发现,AdC6-htertE1A-ΔE3(CSF_(2))能够感染不同的肿瘤细胞,并稳定表达外源基因CSF_(2)。CCK8实验结果表明AdC6-htertE1A-ΔE3和AdC6-htertE1A-ΔE3(CSF_(2))对RD、SW-620、HeLa、Huh7、RM-1与MC-38等多种肿瘤细胞具有明显的杀伤效果;相较于复制缺陷的腺病毒AdC6-ΔE1-ΔE3,感染复数为100 MOI时的AdC6-htertE1A-ΔE3、AdC6-htertE1A-ΔE3(CSF_(2))对肿瘤细胞的杀伤效果极为明显(P<0.05)。Western blot实验证明AdC6-htertE1A-ΔE3和AdC6-htertE1A-ΔE3(CSF_(2))通过激活P53依赖的通路,诱导肿瘤细胞凋亡。在前Objective To construct,with chimpanzee adenovirus serotype 6(AdC6)as the vector,a novel oncolytic adenovirus,enabling it to selectively replicate intratumorally,to test its tumor suppressive effect in vitro and in vivo,and to study its oncolytic mechanism.Methods Based on the AdC6 vector,the human telomerase reverse transcriptase(hTERT)promoter was used to drive the expression of E1A,the adenovirus replication-related gene,and the recombinant oncolytic virus AdC6-htertΔE1A-ΔE3 was thus obtained.The oncolytic virus AdC6-htertE1A-ΔE3(CSF_(2))expressing granulocyte macrophage colony-stimulating factor(GM-CSF/CSF_(2))and replication-deficient adenovirus AdC6-ΔE1-ΔE3 were constructed by homologous recombination,respectively.The recombinant adenovirus was packaged in HEK293 cells,purified and then identified with restriction enzyme digestion.Different types of tumor cells,including RD,SW-620,HeLa,Huh7,RM-1 and MC-38 were infected with the three adenoviruses.Twenty-four hours after infection,Western blot was used to determine the expression of CSF_(2) 24 hours after infection.CCK8 assay was used to determine the survival rate of tumor cells 72 hours after infection.HeLa cells were infected with the three adenoviruses,and the expression levels of apoptosis signaling pathway proteins were examined with Western blot at 12 h,24 h,and 48 h.C57BL/6 mice were subcutaneously injected with cell suspension containing 1×10^(6) MC38 murine colon cancer cells and RM-1 murine prostate cancer cells to construct two tumor-bearing mice models.The tumor-bearing mice were divided into 4 groups,receiving intratumoral injection of 50μL of PBS,AdC6-ΔE1-ΔE3(1×10^(8) PFU),AdC6-htertE1A-ΔE3(1×10^(8) PFU),and AdC6-htertE1A-ΔE3(CSF_(2))(1×10^(8) PFU),respectively.When the tumor size of PBS group reached 2500 mm^(3),all the mice were sacrificed and the tumor tissue was collected for TUNEL staining.Then,apoptosis-positive cells were observed and counted under a microscope.Results Restriction digestion revealed that the oncolytic virus

关 键 词：黑猩猩腺病毒6型 溶瘤病毒 端粒酶逆转录酶启动子 

分 类 号：R73-36[医药卫生—肿瘤]