重组沙门菌SGN1对小鼠黑色素瘤的治疗效果及其作用机制

Therapeutic effect of recombinant Salmonella SGN1 on melanoma in mice and its mechanism of action

目的探讨重组沙门菌SGN1对小鼠黑色素瘤的治疗效果及其相关作用机制,为SGN1临床治疗黑色素瘤提供参考。方法将重组沙门菌SGN1及其对照菌VNP-V与小鼠黑色素瘤细胞B16F10共培养,细胞计数法检测SGN1对B16F10细胞体外增殖的影响;构建B16F10小鼠黑色素瘤皮下移植瘤模型,单次瘤内注射SGN1、VNP-V、PBS(对照),每组5只,观察SGN1对肿瘤生长的抑制作用;HE染色观察小鼠肿瘤组织病理变化;平板计数观察SGN1在小鼠肿瘤组织内分布;流式细胞术检测肿瘤浸润T淋巴细胞比例;免疫荧光和免疫组化染色法检测T细胞标志物CD3的表达。结果与对照组相比,重组沙门菌SGN1可显著抑制小鼠黑素色素瘤B16F10细胞体外增殖(t=6.935,P<0.01);在荷瘤小鼠体内,SGN1具有肿瘤靶向性,并显著抑制B16F10小鼠黑色素瘤皮下移植瘤的生长(t=7.566,P<0.001);HE染色结果显示,SGN1可显著诱导小鼠黑色素瘤皮下移植瘤细胞坏死;免疫荧光、免疫组化和流式细胞术结果显示,SGN1可明显增加肿瘤浸润CD3^(+)T细胞(t分别为11.91、8.873、5.300,P均<0.01)。结论重组沙门菌SGN1可显著抑制小鼠黑色素瘤细胞B16F10的增殖,且通过促进肿瘤细胞坏死及增加CD3^(+)肿瘤浸润T细胞抑制黑色素瘤,为SGN1临床治疗黑色素瘤提供了依据。

Objective To investigate the therapeutic effect of recombinant Salmonella SGN1 on murine melanoma model and the action mechanism,and to provide reference for clinical treatment of melanoma with SGN1.Methods The recombinant Salmonella SGN1 and control strain VNP-V were co-cultured with mouse melanoma B16F10 cells,and the effect of SGN1 on the proliferation of B16F10 cells in vitro was detected by cell counting method.The B16F10 subcutaneous tumor transplantation model in mice was constructed.SGN1,VNP-V and PBS(control group)were injected into the tumor at a single dose,with five mice in each group,and the inhibitory effect of SGN1 on tumor growth was observed.The pathological changes of tumor tissues in mice were observed by HE staining.The distribution of SGN1 in tumor tissues was observed by plate counting.Flow cytometry was used to evaluate the proportion of infiltrating T lymphocyte subsets.The expression of T-cell marker CD3 was detected by immunofluorescence and immunohistochemical staining.Results Compared with the control group,recombinant Salmonella SGN1 significantly inhibited the proliferation of mouse melanoma B16F10 cells in vitro(t=6.935,P<0.01).In tumor-bearing mice,SGN1 was tumor-targeted and significantly inhibited the growth of B16F10 subcutaneous transplanted melanoma in mice(t=7.566,P<0.001).HE staining showed that SGN1 significantly induced the necrosis of subcutaneous transplanted melanoma cells in mice.The results of immunofluorescence,immunohistochemistry and flow cytometry confirmed that SGN1 significantly increased tumor-infiltrating CD3~+T-cells(t=11.91,8.873 and 5.300,respectively,each P<0.01).Conclusion The recombinant Salmonella SGN1 can significantly inhibit the proliferation of B16F10 cells and exert anti-tumor effects by promoting tumor cell necrosis and elevating CD3~+tumor-infiltrating T-cells,providing a theoretical basis for the clinical treatment of melanoma with SGN1.