水疱性口炎病毒对小鼠乳腺癌4T1细胞荷瘤小鼠抗肿瘤免疫、移植瘤生长与肺转移的影响  

Effects of vesicular stomatitis virus on anti-tumour immunity,growth of xenografts,and lung metastasis in mouse mammary carcinoma 4T1 cells tumor-bearing mice

作　　者：李玉迁 徐庆胜 魏洪 王皞[4] 王硕石 蒋立娜 袁心怡 LI Yuqian;XU Qingsheng;WEI Hong;WANG Hao;WANG Shuoshi;JIANG Lina;YUAN Xinyi(Department of Microbiology,Basic Medical College,Guizhou Medical University,Guiyang 550025,Guizhou,China;National Experimental Teaching Center for Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;School of Clinical Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;Emergency surgery of Guizhou Provincial People's Hospital,Guiyang 550005,Guizhou,China;Experimental Center of Hongyan District,the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang 550003,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院微生物学教研室,贵州贵阳550025 [2]贵州医科大学基础医学国家级实验教学中心,贵州贵阳550025 [3]贵州医科大学临床医学院,贵州贵阳550025 [4]贵州省人民医院急诊外科,贵州贵阳550005 [5]贵州中医药大学第二附属医院红岩院区中心实验室,贵州贵阳550003

出　　处：《中国肿瘤生物治疗杂志》2024年第5期452-461,共10页Chinese Journal of Cancer Biotherapy

基　　金：贵州省卫生健康委-科学技术基金(No.gzwkj2023-280);贵州省科技计划项目[No.黔科合基础-ZK(2024)一般125]。

摘　　要：目的:探究野生型水疱性口炎病毒印第安纳株(VSV-IN)对小鼠三阴性乳腺癌4T1细胞移植模型小鼠的免疫调节及肿瘤转移的影响。方法:VSV以MOI=1、MOI=10、MOI=100感染4T1细胞12、24、48 h后,CCK-8法检测4T1细胞死亡率,划痕愈合实验检测细胞迁移能力,qPCR检测细胞中E-cadherin、MMP-2、MMP-9 mRNA的表达。于雌性BALB/c小鼠脂肪垫接种1×10^(6)个/mL的4T1细胞0.1 mL,构建4T1细胞荷瘤小鼠模型,待小鼠肿瘤体积达200 mm3,分别向移植瘤内注射PBS、紫杉醇(TAX)(15 mg/kg)、VSV-IN(1×10^(6)pfu/只),每周1次。给药4次后,处死小鼠、剥离完整移植瘤组织,测量肿瘤体积及质量,肺组织病理切片经H-E染色后观察肿瘤肺部转移结节,流式细胞术检测脾组织中T细胞亚群比例,ELISA法检测小鼠血清IL-6及TNF-α水平,利用GEPIA在线分析乳腺肿中迁移相关蛋白mRNA的表达,免疫组化法检测肿瘤中MMP-2、MMP-9与E-cadherin的表达,利用蛋白-蛋白对接技术预测VSV-IN的G蛋白、M蛋白与ERK2、E-cadherin的亲和力。结果:经MOI=10、100的VSV-IN处理48 h后,4T1细胞死亡率显著高于对照组(均P<0.01);与对照组相比,VSV-IN组(MOI=10)细胞迁移率明显降低(P<0.01),MMP-9 mRNA的相对表达量明显降低(P<0.05);与对照组小鼠相比,VSV-IN组移植瘤生长较对照组减缓且终点体积显著减小(P<0.05),VSV-IN组小鼠肺转移结节数量显著减少[(12.86±1.86)vs(24±3.67)个,P<0.01],脾内CD4^(+)T、CD8^(+)T细胞比例显著升高(均P<0.05),血清TNF-α、IL-6含量显著升高(均P<0.01);GEPIA分析发现在乳腺癌中E-cadherin、MMP-9表达水平均高于癌旁组织(P<0.05);VSV-IN组小鼠肿瘤细胞内MMP-9表达明显低于对照组(P<0.05);VSV-IN的G、M蛋白与ERK2的结合自由能分别为–11.7 kcal/mol、–6.4 kcal/mol。结论:野生型VSV-IN可抑制4T1细胞荷瘤小鼠的移植瘤生长及转移,这可能与其促进抗肿瘤免疫及调控迁移相关蛋白表达有关。Objective:To investigate the effects of wild-type vesicular stomatitis virus strain(VSV-IN)on immunomodulation and tumour metastasis in mouse triple negative breast cancer(TNBC)4T1 cell transplantation model mice.Methods:After VSV-IN infected 4T1 cells with MOI=1,MOI=10 and MOI=100 for 12,24 and 48 h,4T1 cell mortality was detected by CCK-8 method,migration ability by scratch healing assay,and the expressions of E-cadherin,MMP-2 and MMP-9 mRNA by qPCR.The fat pads of female BALB/c mice were inoculated with 0.1 mL of 4T1 cells with a cell density of 1×10^(6)cells/mL to construct a 4T1 cell-loaded mouse model.When the tumour volume reached 200 mm3,the mice were injected intratumorally with PBS,taxol(TAX)(15 mg/kg),and VSV(1×10^(6)pfu)once a week,respectively.After 4 administrations,mice were executed,stripped of intact grafted tumour tissues,and tumour volume and mass were measured.Histopathological sections of the lungs were stained with H-E,and tumour metastatic nodules of the lungs were observed.The proportion of T-cell subpopulations in the spleen was detected by flow cytometry.The levels of serum IL-6 and TNF-αwere detected by ELISA.The expression levels of migration-related proteins in mammary gland tumours were analysed by using the online analysis of GEPIA.The expressions of MMP-2,MMP-9 and E-cadherin in mouse tumours were detected by immunohistochemistry,and the affinity of G and M proteins of VSV-IN and ERK2 and E-cadherin was predicted by protein-protein docking technology.Results:The mortality rate of 4T1 cells after 48 hours of VSV treatment at MOI=10 and 100 were significantly higher than that of the control group(P<0.01).Compared with that of the control group,the cell migration rate of the VSV IN group(MOI=10)was significantly lower(P<0.01),and the relative expression of MMP-9 mRNA was significantly lower(P<0.05).Compared with those in the mice of the control group,transplanted tumours in the mice of the VSV-IN group grew more slowly,and their endpoint volume was significantly reduced(P<0.05).The

关 键 词：野生型水疱性口炎病毒 三阴性乳腺癌 4T1细胞 迁移 蛋白-蛋白对接 

分 类 号：R737.9[医药卫生—肿瘤] R730.54[医药卫生—临床医学]