复方肠泰诱导结肠癌细胞自噬活化巨噬细胞的研究

发布时间：2018-06-21 01:19

本文选题：结肠癌 + 复方肠泰　；参考：《南京中医药大学》2016年硕士论文

【摘要】：目的：通过观察复方肠泰诱导小鼠结肠癌CT26.WT细胞分泌的自噬体对巨噬细胞RAW264.7活化的影响,探讨复方肠泰诱导肿瘤细胞的自噬与抗肿瘤免疫之间的内在关系,从肿瘤免疫学原理来阐明复方肠泰的抗肿瘤作用机制,为中药在抗肿瘤免疫的作用提供实验依据和理论基础。方法：1、复方肠泰诱导小鼠结肠癌CT26.WT细胞自噬的研究利用MTT比色法观察不同浓度复方肠泰对CT26.WT细胞增殖的影响,明确最适给药浓度；Western Blot检测不同浓度复方肠泰作用于CT26.WT细胞后自噬标志蛋白LC3的表达；采用脂质体转染法构建eGFP-LC3 CT26.WT细胞株,复方肠泰干预后荧光显微镜下观察自噬标志蛋白LC3的表达及分布。2、复方肠泰诱导小鼠结肠癌CT26.WT细胞分泌的自噬体的提取与鉴定显微镜观察复方肠泰处理CT26.WT细胞48h后细胞形态的变化,利用专利技术提取囊泡类物质,通过透射电镜观察细胞的超微结构变化、电镜及Western Blot检测自噬标志蛋白LC3的表达以鉴定所提物质是否为自噬体。3、小鼠巨噬细胞RAW264.7对诱导分泌的自噬体的识别与摄取以CFSE标记所提取的自噬体,将其加入RAW264.7细胞(6 μg/ml终浓度)培养48h,流式细胞术和激光共聚焦观察巨噬细胞RAW264.7对自噬体的识别和摄取。4、诱导分泌的自噬体活化小鼠巨噬细胞RAW264.7的研究将提取的自噬体作用于巨噬细胞RAW264.7 (6 μg/ml终浓度),流式细胞术检测自噬体对RAW264.7细胞膜表面共刺激分子CD80、CD40的表达；ELISA检测自噬体刺激巨噬细胞培养上清中的细胞因子TNF-a和IL-6分泌量；qPCR检测巨噬细胞活化的相关细胞因子TNF-α、IL-6、MCP-1、IL-1β的mRNA的转录水平。结果：1、MTT结果显示低浓度复方肠泰作用结肠癌CT26.WT细胞48h,细胞活性无显著变化,无明显的细胞毒性作用,高浓度复方肠泰对CT26.WT细胞有明显的抑制作用,呈剂量依赖性。复方肠泰处理转染了绿色荧光蛋白(EGFP)融合了自噬标记蛋白LC3的CT26.WT细胞后,阴性对照组中可见绿色荧光均匀分布于细胞胞浆内,而加入不同浓度的复方肠泰处理后可见胞浆内出现绿色荧光点,代表着自噬体的形成增多。Western Blot检测结果显示自噬标记蛋白LC3II的表达随着复方肠泰浓度的增加而表达量升高,阳性对照雷帕霉素组亦出现同样现象,表明复方肠泰作用于CT26.WT细胞后可诱导其发生自噬,并形成自噬体。2、显微镜下观察复方肠泰处理CT26.WT细胞48h后形态出现显著变化,细胞内出现大量囊泡样颗粒,随着浓度的增加,囊泡逐渐增多增大。透射电镜进一步证实复方肠泰干预结肠癌CT26.WT细胞后有大量具有双层膜结构的自噬泡形成。利用专利技术提取细胞内的囊泡样物质后,经电镜观察其结构、大小、Western Blot鉴定结果显示所提取的囊泡类物质中含有大量LC3 II,证实其为肿瘤细胞分泌的自噬体。3、以CFSE标记所提取的自噬体刺激RAW264.7细胞48h后,流式细胞术和激光共聚焦观察结果显示自噬体能够被巨噬细胞有效的识别并摄取。4、将所提取的自噬体作用RAW264.7细胞48h之后,可显著上调巨噬细胞表面的共刺激分子CD80及CD40的表达,增加巨噬细胞培养基上清中的TNF-α、IL-6细胞因子的分泌量,升高巨噬细胞活化的相关细胞因子TNF-α、IL-6、MCP-1、IL-1β的mRNA的转录水平。结论：复方肠泰作用于结肠癌CT26.WT细胞后可诱导肿瘤细胞发生自噬,分泌的自噬体能够被巨噬细胞有效识别并摄取,自噬体作用于巨噬细胞后可显著上调其表面的共刺激分子CD80及CD40的表达,增加培养上清中细胞因子TNF-α、IL-6的分泌量,且提高巨噬细胞活化的相关细胞因子TNF-α、IL-6、MCP-1、IL-1β的mRNA的转录水平,表明复方肠泰诱导结肠癌细胞产生的自噬体可激活巨噬细胞,并诱导其向抗肿瘤亚型-M1型活化,增强其杀伤肿瘤的能力。综合以上结果,复方肠泰对结肠癌的抗肿瘤作用与其诱导肿瘤细胞自噬分泌的自噬体活化巨噬细胞,促进抗肿瘤免疫应答相关。
[Abstract]:Objective: To investigate the effect of autophagic on the activation of RAW264.7 in mouse colon cancer CT26.WT cells by observing the effect of compound intestinal Thai on the activation of macrophage, and to explore the intrinsic relationship between autophagy and anti-tumor immunity induced by compound intestinal Thailand, and to clarify the anti tumor mechanism of compound intestinal Thailand from the principle of tumor immunology to prevent swelling of the Chinese medicine. The role of tumor immunity provides experimental basis and theoretical basis. Methods: 1, compound intestinal Thailand induced autophagy of mouse colon cancer CT26.WT cells by using MTT colorimetry to observe the effect of different concentrations of compound intestinal Thailand on the proliferation of CT26.WT cells, and to determine the optimum concentration of drug delivery; Western Blot was used to detect the effect of different concentrations of compound intestinal Thailand on CT26.WT cells. The expression of autophagy marker protein LC3, eGFP-LC3 CT26.WT cell line was constructed by liposome transfection, the expression and distribution of autophagic marker protein LC3 was observed and.2 was observed under the fluorescent microscope with compound intestinal Thailand. The extraction and identification of autophagic in mouse colon cancer CT26.WT cells were induced by compound intestinal Thai, and the identification microscope was used to treat CT26.WT in compound intestinal Thai. The changes in cell morphology after 48h were used to extract the vesicles by patented technology. The ultrastructural changes of cells were observed by transmission electron microscopy. The expression of autophagic protein LC3 was detected by electron microscopy and Western Blot to identify whether the extracts were autophagic.3, and the identification and uptake of the autophagic bodies induced by RAW264.7 in mouse macrophages were identified. The autophago extracted by CFSE markers was added to the RAW264.7 cell (6 micron g/ml final concentration) to cultivate 48h. Flow cytometry and laser confocal microscopy were used to identify and absorb the macrophage RAW264.7 to the autophagic body and uptake.4. The autophagic activated mouse macrophage RAW264.7 was induced by the induced autophagic activation of the macrophage RAW264.7 (6). G/ml terminal concentration), flow cytometry was used to detect the expression of CO stimulatory molecules CD80, CD40 on the membrane surface of RAW264.7 cells by flow cytometry; ELISA was used to detect the cytokine TNF-a and IL-6 secretion in the supernatant of macrophage culture by ELISA; qPCR detected the transcriptional level of macrophage activation related cytokines, TNF- alpha, IL-6, MCP-1, IL-1 beta. Results: 1, MTT results showed that there was no significant change in cell activity of colon cancer CT26.WT cell 48h in low concentration compound intestinal Thai colon cancer cell, no obvious cytotoxicity, high concentration of compound intestinal Thailand had obvious inhibitory effect on CT26.WT cells, and was dose-dependent. Compound intestinal Thai treatment was transfected with green fluorescent protein (EGFP) fusion of autophagy protein LC After 3 CT26.WT cells, the green fluorescence was evenly distributed in the cytoplasm of the negative control group, and the green fluorescence point appeared in the cytoplasm after the addition of different concentrations of compound intestinal Thai, representing the increase of the autophagic body formation and the.Western Blot detection results showed that the expression of autophagy marked egg white LC3II was increased with the increase of the concentration of compound intestinal Thailand. The same phenomenon was found in the positive control group of rapamycin, which showed that compound intestinal Thai could induce autophagy after CT26.WT cells and form autophagic.2. Under microscope, the morphology of CT26.WT cells in compound intestinal Thai treated CT26.WT cells changed significantly, and a large number of vesicle like particles appeared in the cells. With the increase of concentration, the cyst was increased. A number of vesicles increased gradually. Transmission electron microscopy further confirmed that a large number of autophagic vacuoles with double membrane structure were formed after the intervention of compound intestinal Thailand in colon cancer CT26.WT cells. The structure, size, and Western Blot identification of the cysts in the cell were observed by the patent technology. The amount of LC3 II was confirmed to be the autophagosin.3 secreted by the tumor cells. The results of flow cytometry and laser confocal observation showed that the autophagosus was able to be effectively identified and absorbed by macrophages by CFSE labelled autophagosbodies. The autophagosin could be significantly increased by the action of the extracted autophagosin as a RAW264.7 cell 48H. The expression of costimulatory molecules CD80 and CD40 on the cell surface increases the amount of TNF- a, IL-6 cytokine secretion in the supernatant of macrophage culture medium, and increases the transcriptional level of TNF- alpha, IL-6, MCP-1, and IL-1 beta of macrophage activation related cytokines. Conclusion: Compound intestinal Thailand is used to induce tumor cells in colon cancer CT26.WT cells. Autophagic autophagic can be effectively identified and absorbed by macrophages. Autophagic can increase the expression of CO stimulator CD80 and CD40 on the surface of the macrophage, increase the cell factor TNF- a, IL-6 secretion in the culture supernatant, and increase the cell factor related to macrophage activation, TNF- alpha, IL-6, MCP-1, IL-1 beta. The transcriptional level of mRNA indicates that the autophagy produced by compound intestinal Thailand induces macrophages to activate macrophages and induces its activation to the antitumor subtype -M1, and enhances its ability to kill the tumor. Cell, promoting the anti-tumor immune response.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R273

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