| ObjectiveBy observing the effect of autophagosomes secreted by murine CT26.WT colon cancer cells induced by FFCT on the activation of macrophages RAW264.7 cells, to explore the internal relationship between the autophagy of tumor cell induced by FFCT and anti-tumor Immunity, clarify the anti-tumor mechanism of FFCT based on immune principle, provide experimental evidence and theoretical basis for Chinese herbal medicine in anti-tumor immunity.Methods1. The research on the autophagy of murine CT-26 colon cancer cells induced by FFCTTo detect the effect of different concentration FFCT on the proliferation of murine CT-26 colon cancer cells by MTT and define the optimum concentrations. To detect the expression of different dosage of FFCT on the impact of autophagy marker, LC3 protein by Western Blot. Construct eGFP-LC3 CT26.WT cell line by liposome transfection, Immunofluorescene staining was used to observe the expression and distribution of autophagy marker, LC3 after FFCT intervene murine CT-26 colon cancer cells.2. The extraction and identification of autophagosome secreted by murine CT-26 colon cancer cells induced by FFCTCellular morphological changes were observed under inverted phase contrast microscope and Transmission Election Microscopy was employed to further examine the changes of ultrastructure. Identify the exerct by Western Blot through the expression of autophagy marker protein, LC3.3. The ability of RAW264.7 macrophage cells to identify and uptake the autophagosome extracted.Labeled the extracted autophagosome with CFSE, injected into RAW264.7 macrophage cells for 48h, flow cytometry and Laser Scan Confocal Microscope were used to observe the ability of RAW264.7 macrophage cells to identify and uptake the autophagosome.4. The study of the RAW264.7 cells activated by the extracted autophagosomeAfter the extracted autophagosome act directly on RAW264.7 macrophage cells, flow cytometry were used to detect the expression level of the surface of CD80 and CD40, ELIS A to analyzed the expression of TNF-a and IL-6, qPCR to examin the mRNA expression of TNF-α IL-6、MCP-1、IL-1β.Results1. MTT assay results showed that low concentration FFCT have no direct toxic effects on CT26.WT cells, cellular activity has no significant change. While high concentration remarkably inhibited the proliferation of CT26.WT cells in dose dependent manner. Manner. LC3 protein of CT26.WT cells in control group was homogeneous distribution in cytoplasm, but in FFCT group, there was acyivation and degradation of autophagy protein LC3; Western Blot assay showed that after treatment with FFCT, type cytoplasm LC3(LC3-I) degraded into type (autophagy) film (LC3-Ⅱ),indicated that FFCT can induce CT26.WT cells autophagy.2. The remarkable morphological changes were observed by inverted phase contrast microscope. It was visible that there were abundant cytoplasmic vacuoles of varying sizes in CT 26.WT cells treated with FFCT. Furthermore, with the increase of concentration, vacuolization in cytoplasm progressively become larger and denser.What is important, electron microscopy further demonstrated formation of abundant autophagic vacuoles in CT26.WT cells followed FFCT treated. Identification result by Western Blot showed that vacuoles contain extensive LC3-II, which confirmed that the vacuoles extracted were autophagosome3. Flow cytometry and Laser Scan Confocal Microscope showed that RAW264.7 macrophage cells can effectively identify and uptake the autophagosome which were Labeled with CFSE4. Result showed that After the extracted autophagosome act directly on RAW264.7 macrophage cells can visibly promote the cell surface molecular expression level of CD80 and CD40, meanwhile, the concentration of inflammatory cytokines in cell culture supernatant such as TNF-a and IL-6 increased, the mRNA expression of TNF-α、IL-6、MCP-1、IL-1β was remarkably enhanced.ConclusionsFFCT act on murine CT-26 colon cancer cells can induce autophagy and secret autophagosomes, which can be identified and uptake by RAW264.7 macrophage cells, then visibly promote the cell surface molecular expression level of CD80 and CD40, meanwhile, increase the realease of inflammatory cytokines such as TNF-a and IL-6, remarkably enhance the mRNA expression of TNF-α、IL-6、MCP-1、IL-1β. It showed that autophagosome can activate the macrophage cells,and induces the activation of anti-Tumor subtype-M1, strengthen immune system, further enhanced The ability of phagocytosis to anti tumor.Based on the above results,the antitumor effect of FFCT acted on colon cancer has close association with autophagosomes generated by its induction of tumor cell, which can activate macrophages, promote the antitumor immune response. |
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