六味地黄丸增效小鼠黑色素瘤HSV-tk/GCV自杀基因疗法的免疫机制

Immune Mechanism of Liuwei Dihuang Wan to Synergize HSV-tk/GCV Suicide Gene Therapy for Melanoma in Mice

目的明确六味地黄丸是否增强单纯疱疹病毒核苷激酶/更昔洛韦(HSV-tk/GCV)自杀基因疗法对小鼠黑色素瘤的免疫杀伤效果,并对其相关机制进行探索。方法采用Western Blot实验、免疫荧光实验检测六味地黄丸对缝隙连接蛋白43(Cx43)表达水平及膜定位的影响;通过荧光黄染料、钙黄绿素荧光传输实验检测六味地黄丸对小鼠黑色素瘤细胞B16(B16细胞)缝隙连接通讯(GJIC)功能的影响;PI/Hoechst染色实验检测CD8+T细胞对B16细胞的免疫杀伤效果;CD8+T细胞激活实验观察GJIC在六味地黄丸增强对小鼠黑色素瘤免疫杀伤中的作用。结果自杀基因疗法联合六味地黄丸治疗较单独应用自杀基因疗法能更显著地抑制肿瘤生长;Western Blot及免疫荧光实验显示六味地黄丸组Cx43表达明显上调,荧光传输实验显示六味地黄丸组B16细胞GJIC功能显著增强;PI/Hoechst染色实验显示,六味地黄丸组CD8+T细胞对B16细胞的免疫杀伤效果更加显著,阻断Cx43后,对B16细胞的免疫杀伤效果则被逆转,同时CD8+T细胞的激活也被减弱。结论六味地黄丸可通过上调B16细胞中Cx43的表达,改善GJIC功能,促进肿瘤抗原的交叉提呈,更强地激活CD8+T细胞以增强其对肿瘤细胞的免疫杀伤效果。

Objective To determine whether Liuwei Dihuang Wan(LWDHW)can actually enhance the immune killing effect of suicide gene therapy on melanoma of mice in vivo and in vitro,and explore the related mechanisms.Methods Western Blot and immunofluorescence assays were used to detect the effect of LWDHW on Cx43 expression level and membrane positioning.The Lucifer Yellow staining assay was used to determine the effect of LWDHW on the GJIC function of B16 cells.PI/Hoechst staining was used to measure the immune killing effect of CD8+T cells on B16 melanoma cells.CD8+T cell activation was determined by CD69 staining followed with flow cytometry detection.Results Suicide gene system combined with LWDHW treatment can inhibit tumor growth more significantly than HSV-tk/GCV system alone.Western Blot and immunofluorescence results showed that the expression of Cx43 in the LWDHW group was significantly up-regulated,and the Lucifer Yellow staining assay showed that GJIC function of B16 cells in the LWDHW group was significantly enhanced.PI/Hoechst staining showed that the immune killing effect on B16 cells in the LWDHW group was more obvious.After blocking Cx43 by Gap19-TFA,the immune killing on B16 cells was reversed and CD8+T cells activation was also impaired.Conclusion LWDHW improved the GJIC function by increasing the expression of Cx43 in B16 cells,promoting the cross-presentation of tumor antigens,thereby priming T cells more strongly to enhance the immune killing effect on melanoma cells.