[关键词]
[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1×106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(1×106 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。
[Key word]
[Abstract]
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 1×106 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 (1×106 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-α were 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 number of lung metastatic nodules in the mice of the VSV group was significantly less than that of the control group ([12.86±1.86] vs [24±3.67], P<0.01). The proportions of splenic CD4+ T and CD8+ T cells in the VSV group were significantly higher (both P<0.05). The serum TNF-α and IL-6 levels were significantly higher (both P<0.01). GEPIA tool analysis revealed that the expression levels of E-cadherin and MMP-9 were higher in breast cancer tissues than in paracancerous tissues (P<0.05). The expression of MMP-9 in the tumour cells of the mice in the VSV-IN group was significantly lower than that in the control group (P<0.05). The binding free energies of G and M proteins of VSV-IN to ERK2 were -11.7 kcal/mol and -6.4 kcal/mol, respectively. Conclusion: Wild-type VSV-IN inhibits the growth and metastasis of transplanted tumours in 4T1 tumor-bearing mice, which may be related to its promotion of anti-tumour immunity and modulation of the expression of migration-related proteins.
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[基金项目]
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]