摘要
目的探讨柯萨奇病毒和腺病毒受体(coxsackievirus-adenovirus receptor,CAR)在胸腺癌中的表达和溶瘤腺病毒H101抗肿瘤活性之间的关系及其机制研究。方法实时荧光定量聚合酶链反应(RT-qPCR)和Western blot检测胸腺癌组织和细胞中CAR的基因和蛋白质表达量。H101病毒感染Bcap-37、MP59和T1889细胞后,RT-qPCR和半数组织培养感染量(TCID50)法检测细胞中H101的表达及上清中病毒滴度。不同感染复数(MOI)H101感染T1889细胞后,CCK-8法检测各组细胞的增殖活性,流式细胞术检测细胞凋亡率。不同浓度组蛋白去乙酰化酶抑制剂曲古菌素A(Trichostatin A,TSA)处理T1889细胞后检测细胞中CAR的表达量;TSA处理细胞后,感染H101,检测细胞中H101的表达量及病毒滴度,CCK-8法检测细胞活性;TSA处理或TSA+ERK激活剂TBHQ处理细胞后检测T1889细胞中MARK、ERK1/2的磷酸化水平和CAR的蛋白质表达量。结果与癌旁正常组织相比,胸腺癌组织中CAR的基因和蛋白质表达量均显著降低(P<0.01);胸腺癌MP59细胞和T1889细胞中CAR表达量明显低于胸腺上皮细胞(TEC)和Bcap-37细胞(P<0.01),H101表达量及上清中H101滴度明显低于Bcap-37细胞(P<0.01);H101感染细胞48 h后,MP59和T1889细胞活性与Bcap-37细胞相比明显升高,细胞凋亡率明显下降(P<0.01)。不同浓度TSA处理后,T1889细胞中CAR基因和蛋白质水平呈剂量依赖性升高。0.25μmol/L TSA处理T1889细胞后,H101基因表达量和病毒滴度明显升高(P<0.05),MAPK和ERK1/2蛋白的磷酸化水平不断下降,CAR表达不断升高;和TSA处理组相比,TSA+TBHQ处理组细胞内CAR蛋白质表达量明显下降(P<0.01),p-ERK1/2/ERK1/2比值明显升高(P<0.01)。结论TSA通过抑制MARK/ERK1/2通路上调CAR在胸腺癌中的表达,从而增强H101的抗肿瘤活性。
Objective To investigate the expression of coxsackievirus-adenovirus receptor(CAR)in thymic carcinoma and the relationship between CAR and the antitumor activity of oncolytic adenovirus H101.Methods The expression of CAR in thymic carcinoma tissues and cells were detected by RT-qPCR and Western blot.H101 expression and virus titers in Bcap-37,MP59 and T1889 cells after infection were detected by RT-qPCR and 50%tissue culture infectious dose(TCID50).The proliferation activity and apoptosis rates of T1889 cells infected with H101 at different multiplicity of infection(MOI)were detected by CCK-8 and flow cytometry.CAR expression in T1889 cells treated with different concentrations of trichostatin A(TSA),a histone deacetylase inhibitor,was detected.H101 expression and virus titers in the TSA-treated and H101-infected cells were detected.Cell activity was detected by CCK-8.The phosphorylation levels of MARK and ERK1/2 and the expression of CAR at protein level in TSA-treated or TSA+TBHQ(ERK activator)treated cells were detected.Results CAR expression at both mRNA and protein levels were significantly lower in thymic carcinoma tissues than in adjacent normal tissues(P<0.01),and lower in MP59 and T1889 cells than in thymic epithelial cells(TEC)and Bcap-37 cells(P<0.01).H101 expression in MP59 and T1889 cells and the titers of H101 in culture supernatants were significantly lower than those in Bcap-37 cells(P<0.01).Compared with Bcap-37 cell,the activity of MP59 and T1889 cells was significantly increased and the apoptosis rates were significantly decreased 48 h after H101 infection(P<0.01).The expression of CAR at both mRNA and protein levels in T1889 cells treated with different concentrations of TSA increased in a dose-dependent manner.When T1889 cells were treated with 0.25μmol/L of TSA,the expression of H101 at mRNA level and H101 titers were significantly increased(P<0.05);the phosphorylation levels of MAPK and ERK1/2 proteins were continuously decreased;the expression of CAR was continuously increased.Compared wi
作者
何占锋
王伟
郑天亮
刘东雷
杨洋
朱登彦
吴恺
王丽萍
赵松
He Zhanfeng;Wang Wei;Zheng Tianliang;Liu Donglei;Yang Yang;Zhu Dengyan;Wu Kai;Wang Liping;Zhao Song(Department of Thoracic Surgery,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China;Henan Medical Association,Zhengzhou 450003,China;Department of Clinical Medicine Zhengzhou University,Zhengzhou 450052,China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2020年第8期628-634,共7页
Chinese Journal of Microbiology and Immunology
