本文選題：結(jié)腸癌 + 復(fù)方腸泰�。� 參考：《南京中醫(yī)藥大學(xué)》2016年碩士論文

【摘要】：目的：通過觀察復(fù)方腸泰誘導(dǎo)小鼠結(jié)腸癌CT26.WT細(xì)胞分泌的自噬體對巨噬細(xì)胞RAW264.7活化的影響,探討復(fù)方腸泰誘導(dǎo)腫瘤細(xì)胞的自噬與抗腫瘤免疫之間的內(nèi)在關(guān)系,從腫瘤免疫學(xué)原理來闡明復(fù)方腸泰的抗腫瘤作用機(jī)制,為中藥在抗腫瘤免疫的作用提供實驗依據(jù)和理論基礎(chǔ)。方法：1、復(fù)方腸泰誘導(dǎo)小鼠結(jié)腸癌CT26.WT細(xì)胞自噬的研究利用MTT比色法觀察不同濃度復(fù)方腸泰對CT26.WT細(xì)胞增殖的影響,明確最適給藥濃度；Western Blot檢測不同濃度復(fù)方腸泰作用于CT26.WT細(xì)胞后自噬標(biāo)志蛋白LC3的表達(dá)；采用脂質(zhì)體轉(zhuǎn)染法構(gòu)建eGFP-LC3 CT26.WT細(xì)胞株,復(fù)方腸泰干預(yù)后熒光顯微鏡下觀察自噬標(biāo)志蛋白LC3的表達(dá)及分布。2、復(fù)方腸泰誘導(dǎo)小鼠結(jié)腸癌CT26.WT細(xì)胞分泌的自噬體的提取與鑒定顯微鏡觀察復(fù)方腸泰處理CT26.WT細(xì)胞48h后細(xì)胞形態(tài)的變化,利用專利技術(shù)提取囊泡類物質(zhì),通過透射電鏡觀察細(xì)胞的超微結(jié)構(gòu)變化、電鏡及Western Blot檢測自噬標(biāo)志蛋白LC3的表達(dá)以鑒定所提物質(zhì)是否為自噬體。3、小鼠巨噬細(xì)胞RAW264.7對誘導(dǎo)分泌的自噬體的識別與攝取以CFSE標(biāo)記所提取的自噬體,將其加入RAW264.7細(xì)胞(6 μg/ml終濃度)培養(yǎng)48h,流式細(xì)胞術(shù)和激光共聚焦觀察巨噬細(xì)胞RAW264.7對自噬體的識別和攝取。4、誘導(dǎo)分泌的自噬體活化小鼠巨噬細(xì)胞RAW264.7的研究將提取的自噬體作用于巨噬細(xì)胞RAW264.7 (6 μg/ml終濃度),流式細(xì)胞術(shù)檢測自噬體對RAW264.7細(xì)胞膜表面共刺激分子CD80、CD40的表達(dá)；ELISA檢測自噬體刺激巨噬細(xì)胞培養(yǎng)上清中的細(xì)胞因子TNF-a和IL-6分泌量；qPCR檢測巨噬細(xì)胞活化的相關(guān)細(xì)胞因子TNF-α、IL-6、MCP-1、IL-1β的mRNA的轉(zhuǎn)錄水平。結(jié)果：1、MTT結(jié)果顯示低濃度復(fù)方腸泰作用結(jié)腸癌CT26.WT細(xì)胞48h,細(xì)胞活性無顯著變化,無明顯的細(xì)胞毒性作用,高濃度復(fù)方腸泰對CT26.WT細(xì)胞有明顯的抑制作用,呈劑量依賴性。復(fù)方腸泰處理轉(zhuǎn)染了綠色熒光蛋白(EGFP)融合了自噬標(biāo)記蛋白LC3的CT26.WT細(xì)胞后,陰性對照組中可見綠色熒光均勻分布于細(xì)胞胞漿內(nèi),而加入不同濃度的復(fù)方腸泰處理后可見胞漿內(nèi)出現(xiàn)綠色熒光點,代表著自噬體的形成增多。Western Blot檢測結(jié)果顯示自噬標(biāo)記蛋白LC3II的表達(dá)隨著復(fù)方腸泰濃度的增加而表達(dá)量升高,陽性對照雷帕霉素組亦出現(xiàn)同樣現(xiàn)象,表明復(fù)方腸泰作用于CT26.WT細(xì)胞后可誘導(dǎo)其發(fā)生自噬,并形成自噬體。2、顯微鏡下觀察復(fù)方腸泰處理CT26.WT細(xì)胞48h后形態(tài)出現(xiàn)顯著變化,細(xì)胞內(nèi)出現(xiàn)大量囊泡樣顆粒,隨著濃度的增加,囊泡逐漸增多增大。透射電鏡進(jìn)一步證實復(fù)方腸泰干預(yù)結(jié)腸癌CT26.WT細(xì)胞后有大量具有雙層膜結(jié)構(gòu)的自噬泡形成。利用專利技術(shù)提取細(xì)胞內(nèi)的囊泡樣物質(zhì)后,經(jīng)電鏡觀察其結(jié)構(gòu)、大小、Western Blot鑒定結(jié)果顯示所提取的囊泡類物質(zhì)中含有大量LC3 II,證實其為腫瘤細(xì)胞分泌的自噬體。3、以CFSE標(biāo)記所提取的自噬體刺激RAW264.7細(xì)胞48h后,流式細(xì)胞術(shù)和激光共聚焦觀察結(jié)果顯示自噬體能夠被巨噬細(xì)胞有效的識別并攝取。4、將所提取的自噬體作用RAW264.7細(xì)胞48h之后,可顯著上調(diào)巨噬細(xì)胞表面的共刺激分子CD80及CD40的表達(dá),增加巨噬細(xì)胞培養(yǎng)基上清中的TNF-α、IL-6細(xì)胞因子的分泌量,升高巨噬細(xì)胞活化的相關(guān)細(xì)胞因子TNF-α、IL-6、MCP-1、IL-1β的mRNA的轉(zhuǎn)錄水平。結(jié)論：復(fù)方腸泰作用于結(jié)腸癌CT26.WT細(xì)胞后可誘導(dǎo)腫瘤細(xì)胞發(fā)生自噬,分泌的自噬體能夠被巨噬細(xì)胞有效識別并攝取,自噬體作用于巨噬細(xì)胞后可顯著上調(diào)其表面的共刺激分子CD80及CD40的表達(dá),增加培養(yǎng)上清中細(xì)胞因子TNF-α、IL-6的分泌量,且提高巨噬細(xì)胞活化的相關(guān)細(xì)胞因子TNF-α、IL-6、MCP-1、IL-1β的mRNA的轉(zhuǎn)錄水平,表明復(fù)方腸泰誘導(dǎo)結(jié)腸癌細(xì)胞產(chǎn)生的自噬體可激活巨噬細(xì)胞,并誘導(dǎo)其向抗腫瘤亞型-M1型活化,增強(qiáng)其殺傷腫瘤的能力。綜合以上結(jié)果,復(fù)方腸泰對結(jié)腸癌的抗腫瘤作用與其誘導(dǎo)腫瘤細(xì)胞自噬分泌的自噬體活化巨噬細(xì)胞,促進(jìn)抗腫瘤免疫應(yīng)答相關(guān)。
[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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R273

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