六味地黄丸通过干预缝隙连接通讯功能调控树突状细胞抗原交叉提呈能力研究

Study on the Effect of Liuwei Dihuang Pills on Regulating the AntigenCross-Presenting Ability of Dendritic Cells by Interfering withGap Junctional Communication Function

【目的】探讨六味地黄丸是否通过提高缝隙连接通讯功能(GJIC)增强树突状细胞(DC)抗原交叉提呈能力,并对其机制进行相关探索。【方法】采用Western Blot实验、免疫荧光法观察六味地黄丸含药血清对黑色素瘤细胞(B16)缝隙连接蛋白43(Cx43)表达及膜定位的影响;采用钙黄绿素(Ca-AM)/DiI荧光示踪实验观察六味地黄丸含药血清对B16细胞GJIC功能的影响;采用流式细胞术观察GJIC在六味地黄丸含药血清增强DC抗原提呈能力中的作用;采用碘化丙啶(PI)/Hoechst染色实验观察CD8+T淋巴细胞的免疫杀伤效果。【结果】Western Blot及免疫荧光实验结果显示,六味地黄丸含药血清上调Cx43表达;荧光示踪实验证明,六味地黄丸含药血清显著增强B16细胞GJIC功能;流式细胞术分析表明,六味地黄丸含药血清增强DC抗原提呈能力;PI/Hoechst染色结果显示,六味地黄丸含药血清干预B16-OVA后CD8+T细胞的免疫杀伤效果更加显著。【结论】六味地黄丸通过上调黑色素瘤细胞Cx43表达,改善GJIC功能,进而增强DC的交叉提呈能力,从而激活更强的CD8+T细胞免疫杀伤反应。

Objective To investigate whether Liuwei Dihuang Pills enhances the antigen cross-presenting ability of dendritic cell(DC)by increasing gap junctional intercellular communication(GJIC),and to explore the mechanisms involved.Methods Western Blot and immunofluorescence were used to observe the effects of Liuwei Dihuang Pillscontaining serum on the expression and membrane localisation of gap junction protein connexin43(Cx43)in mouse melanoma cells(B16);Calcein-AM/DiI fluorescence tracer assay was used to observe the effects of Liuwei Dihuang Pills-containing serum on the function of GJIC in B16 cells;flow cytometry was used to observe the role of GJIC in the enhancement of DC antigen presenting ability by Liuwei Dihuang Pills-containing serum;and propidium iodide(PI)/Hoechst staining assay was used to observe the immunocidal effect of CD8+T-lymphocytes.Results Western Blot and immunofluorescence experiments showed that Liuwei Dihuang Pills-containing serum led to the up-regulation of Cx43 expression;fluorescence tracer experiments proved that the GJIC function of B16 cells was significantly enhanced by Liuwei Dihuang Pills-containing serum;flow cytometry analyses showed thatthe DC antigen-presenting ability was enhanced by Liuwei Dihuang Pills-containing serum;and the results of PI/Hoechst staining showed that the immuno-killing effect of CD8+ T-cells was more significant after the intervention ofLiuwei Dihuang Pills-containing serum in B16-OVA. Conclusion Liuwei Dihuang Pills improve the GJIC functionby up-regulating the Cx43 expression of melanoma cells,and then enhance the cross-presenting ability of DCsthus activating stronger CD8+ T-cell immunocidal responses.