海洋微生物环肽Ilamycin E抗三阴性乳腺癌的作用及机制研究

发布时间：2018-06-20 09:09

本文选题：天然化合产物 + Ilamycin　；参考：《南方医科大学》2017年硕士论文

【摘要】：意义乳腺癌是世界范围内女性最为常见的恶性肿瘤,在女性因癌症致死原因中占据主导地位。三阴性乳腺癌(TNBC)定义为缺乏雌激素受体(ER)、孕激素受体(PR)以及人表皮生长因子受体2(HER2)基因的特殊乳腺癌亚型,占乳腺癌全部类型15-20%。TNBC具有高风险的转移与复发率,目前仍缺乏有效的治疗措施。天然化合产物Ilamycin家族,来源于海洋微生物次级代谢产物,其细胞毒活性从来没有被报道过。有初步研究结果显示,化合物Ilamycin具有抗肿瘤活性,但仍需进一步深入研究。方法1.SRB法检测细胞存活率,反映化合物Ilamycin E,F对乳腺癌细胞存活的影响。使用不同浓度 Ilamycin E,F 在 HCC 1806,HCC 1937,MDA-MB-468,MDA-MB-231,T47D,MCF7,SKBR3,BT474,MCF10A 细胞系验证效果,并进一步用SRB的方法测定每株细胞系准确的IC50。最后根据实验需要选出作为研究的代表细胞系,作为后续实验的材料。2.EdU检测细胞增殖为了检测Ilamycin E对细胞增殖的影响,以不同浓度Ilamycin E(0,10,20,30μM)处理 HCC1937 和 MDA-MB-468 细胞 24 小时,以 EPI(0.2μM)作为阳性对照,然后用Click-iT EdU试剂盒进行处理,最后封片拍照,所得结果用ImageJ和IPP软件处理,从每个处理结果中选择3张图片进行统计、处理,所得比值作为最后结果。3.细胞周期试验为了进一步分析Ilamycin E导致细胞存活抑制的主要方式,我们使用流式细胞仪检测化合物对细胞周期进程的影响。用IlamycinE不同浓度(0,10,20,30μM)处理HCC1937,MDA-MB-468细胞24小时,胰酶消化,75%乙醇4℃固定。最后以大约1x106个细胞用100 μl 0.6%NP40+1μl PI+1 μ1 RNase的比例配成体系,重悬细胞。染色处理,AccuriC6(BD)上机分析细胞周期,软件FlowJo分析统计结果。4.细胞凋亡分析为了检测化合物Ilamycin E对细胞凋亡的影响,我们使用Annexin V和PI双染流式细胞方法检测化合物Ilamycin E对细胞凋亡的影响。使用Ilamycin E不同浓度(0,10,20,30μM)处理HCC1937细胞24小时,处理MDA-MB-468细胞36小时,Annexin V和PI双染流式细胞仪上机检测、分析细胞凋亡情况。5.蛋白印迹检测周期和凋亡相关蛋白的改变,用以探索凋亡机制为了探讨细胞周期和凋亡改变的机制,我们做了大量蛋白筛查,使用不同浓度Ilamycin E(0,10,20,30μM)处理48小时或用30μM单浓度处理HCC1937,MDA-MB-468细胞不同时间,加细胞裂解液冰上裂解30min,离心,蛋白定量,然后加4×SDS Loading Buffer,98 ℃ 5min煮样变性,上样,SDS聚丙烯酰胺凝胶电泳分离蛋白,转膜,抗体孵育14小时,二抗孵育2小时,曝光仪检测检测各个目标条带变化情况。使用Photoshop作图软件进行数据处理。6.数据处理为了保证数据的准确性,实验数据进行至少三次独立重复的验证。使用软件SPSS运算t检验进行显著性分析,P值0.05则认为有显著性差异。流式结果使用FlowJo处理,柱形图和线形图使用GraphPad制作。Edu结果使用ImageJ,IPP进行重叠计数。结果1.SRB细胞存活率检测结果显示,化合物Ilamycin E对HCC 1806,HCC 1937,MDA-MB-468,MDA-MB-231,T47D,MCF7,SKBR3,BT474,MCF10A 细胞具有广泛的细胞毒活性,同时化合物Ilamycin F不具有或在所给有效剂量范围内不具有细胞毒活性。化合物IC50检测结果显示,Ilamycin E对细胞有效IC50分布为14.24-49.19μM,三阴性乳腺癌细胞HCC1937细胞系最为敏感,而人永生化乳腺上皮细胞MCF10A细胞系敏感性最差。化合物Ilamycin E与F的结果差异表明Ilamycin家族化合物细胞毒活性具有特定的化学基团,这为化合物的改造指明了方向。2.EdU细胞增殖实验结果显示,化合物Ilamycin E显著抑制三阴性乳腺癌细胞系HCC1937和MDA-MB-468的DNA合成,减少细胞分裂,证明细胞增殖受到抑制。因此,我们初步得出结论,即化合物Ilamycin E导致的细胞存活抑制部分归功于细胞增殖抑制。3.流式细胞周期检测结果显示,随着化合物Ilamycin E有效作用浓度的递增,Ilamycin E显著增加HCC1937和MDA-MB-468两株三阴性乳腺癌细胞G0/G1期细胞比例,并降低细胞S期和G2/M期细胞比例,化合物Ilamycin E有效抑制HCC1937 和 MDA-MB-468 细胞 G1/S 进程。4.流式细胞凋亡实验结果显示,化合物Ilamycin E明显导致HCC1937和MDA-MB-468两株三阴性乳腺癌细胞凋亡比例增加,并且随着化合物Ilamycin E有效作用浓度的增加,细胞凋亡比例逐渐增加。实验结果表明,化合物Ilamycin E导致的细胞存活抑制部分原因归功于诱导细胞凋亡。5.使用蛋白印迹实验经过大量筛查表明,Ilamycin E明显导致三阴性乳腺癌细胞系HCC1937和MDA-MB-468 一系列周期、凋亡相关蛋白的变化。同时,Real-time PCR检测结果显示,化合物Ilamycin E下调KLF5以及上调EGR1的转录。而ERStress信号通路相关蛋白Bip,IRE1α,XIAP,CHOP均有显著增加。实验结果表明,化合物IlamycinE在调控细胞周期、凋亡相关蛋白表达的同时,显著诱导细胞ER Stress信号通路的活化。结论本研究结果显示,Ilamycin家族成员IlamycinE具有潜在的抗三阴性乳腺癌活性。化合物Ilamycin E导致三阴性乳腺癌HCC1937和MDA-MB-468细胞周期相关蛋白Cyclin D1显著下降,而细胞周期蛋白依赖性激酶抑制剂p21、p27显著上升,并引起细胞G1/S期细胞周期进程阻滞。化合物IlamycinE还能有效诱导HCC1937和MDA-MB-468细胞ER stress的产生,并导致细胞凋亡。实验还发现化合物Ilamycin E能有效抑制KLF5转录,同时上调肿瘤抑制因子EGR1的表达。综合结果表明化合物IlamycinE能有效抑制三阴性乳腺癌,这或许将为三阴性乳腺癌的治疗提供更多的可能。
[Abstract]:Significant breast cancer is the most common malignant tumor in women worldwide. It occupies a dominant position in women's cause of cancer death. Three negative breast cancer (TNBC) is defined as a special breast cancer subtype lacking estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) gene, which accounts for all types of 15-20%. of breast cancer. TNBC has a high risk of metastasis and recurrence, and there is still a lack of effective treatment. The Ilamycin family of natural chemical products, derived from the secondary metabolites of marine microorganism, has never been reported on cytotoxic activity. Preliminary results show that compound Ilamycin has antitumor activity, but it still needs further study. The effect of Ilamycin E and F on the survival of breast cancer cells was measured by the method of 1.SRB, and Ilamycin E, F in HCC 1806, HCC 1937, MDA-MB-468, MDA-MB-231, T47D, etc. The test needs to be selected as the representative cell line of the study. As a follow-up experiment material.2.EdU detected cell proliferation in order to detect the effect of Ilamycin E on cell proliferation, HCC1937 and MDA-MB-468 cells were treated with different concentrations of Ilamycin E (0,10,20,30 mu M) for 24 hours, and EPI (0.2 mu M) as positive control, and then into the Click-iT cartridge kit. The results were processed with ImageJ and IPP software. 3 pictures were selected for statistics, processing, and the ratio was used as the final result of.3. cell cycle test in order to further analyze the main way that Ilamycin E resulted in cell survival inhibition. We used flow cytometer to detect compound pairs. The effects of cell cycle process were treated with different concentrations of IlamycinE (0,10,20,30 M) for HCC1937, MDA-MB-468 cells for 24 hours, trypsin digestion, and 75% ethanol at 4 centigrade. Finally, the cell cycle was analyzed by the proportion of about 1x106 cells with 100 L 0.6%NP40+1 u l PI+1 micron 1 RNase. Jo analysis showed that.4. cell apoptosis was analyzed in order to detect the effect of compound Ilamycin E on cell apoptosis. We used Annexin V and PI double stained flow cytometry to detect the effect of Ilamycin E on cell apoptosis. The cells were treated with Ilamycin E (0,10,20,30 muon) for 24 hours and treated for 36 hours. Annexin V and PI double dye flow cytometry were used to detect the cell apoptosis and the changes of.5. blot detection cycle and apoptosis related protein. In order to explore the mechanism of apoptosis mechanism in order to explore the mechanism of cell cycle and apoptosis change, we have done a lot of protein screening, using different concentrations of Ilamycin E (0,10,20,30 mu M) for 48 hours or 30. M single concentration of HCC1937, MDA-MB-468 cells at different time, add cell lysate on ice to crack 30min, centrifugation, protein quantitative, then add 4 x SDS Loading Buffer, 98 5min to cook denaturation, sample, SDS polyacrylamide gel electrophoresis to separate protein, turn film, incubate for 14 hours, two anti incubation for 2 hours, exposure meter detection and detection of the various targets The Photoshop mapping software is used to process data processing.6. data processing to ensure the accuracy of the data, the experimental data is verified at least three times independently. Using the software SPSS to perform the significant analysis of the t test, the P value 0.05 considers the significant difference. The flow results use FlowJo processing, column chart and line shape. GraphPad.Edu results were made using ImageJ and IPP for overlapping counts. Results the results of 1.SRB cell viability test showed that compound Ilamycin E had extensive cytotoxic activity to HCC 1806, HCC 1937, MDA-MB-468, MDA-MB-231, T47D, T47D, etc. The results of IC50 detection showed that the effective IC50 distribution of Ilamycin E to the cells was 14.24-49.19 u M, the HCC1937 cell line of three negative breast cancer cells was most sensitive and the sensitivity of MCF10A cell line of human immortalized mammary epithelial cells was the worst. The difference between Ilamycin E and F showed that Ilamycin familial The cytotoxic activity of the compound has a specific chemical group. The modification of the compound indicates that the.2.EdU cell proliferation test results show that compound Ilamycin E significantly inhibits the DNA synthesis of three negative breast cancer cell lines HCC1937 and MDA-MB-468, reducing cell division and inhibiting cell proliferation. Therefore, we conclude that the cell proliferation is inhibited. The cell survival inhibition caused by compound Ilamycin E was attributed to the cell proliferation inhibition.3. flow cytometric detection results, and with the increase of the effective concentration of the compound Ilamycin E, Ilamycin E significantly increased the ratio of HCC1937 and MDA-MB-468 two negative breast cancer cells G0/ G1 phase cells, and reduced the cell S phase and the duration. Cell ratio, compound Ilamycin E effectively inhibited the.4. flow cytometry of G1/S process in HCC1937 and MDA-MB-468 cells, and the results showed that compound Ilamycin E significantly increased the proportion of apoptosis in HCC1937 and MDA-MB-468 two strains of three negative breast cancer cells, and the cell apoptosis ratio was increased with the increase of the effective concentration of Ilamycin E. The experimental results showed that the cell survival inhibition caused by compound Ilamycin E was due to the induction of cell apoptosis partly due to the induction of apoptosis in.5.. A large number of screening tests showed that Ilamycin E significantly resulted in a series of cycles of HCC1937 and MDA-MB-468 in the three negative breast cancer cell lines, the changes in apoptosis related proteins, and Real-t. The results of IME PCR detection showed that compound Ilamycin E downregulated KLF5 and up regulation of EGR1, while ERStress signaling pathway related proteins Bip, IRE1 alpha, XIAP, CHOP were significantly increased. The experimental results showed that compound IlamycinE was activated by the regulation of cell cycle and expression of apoptosis related proteins. Conclusion the results of this study showed that the Ilamycin family member IlamycinE had a potential anti three negative breast cancer activity. Compound Ilamycin E resulted in a significant decrease in the HCC1937 and MDA-MB-468 cell cycle related protein Cyclin D1 in three negative breast cancers, while the cyclin dependent kinase inhibitor p21, p27 increased significantly, and resulted in the cell G1/S phase. The cell cycle process block. Compound IlamycinE can also effectively induce the production of ER stress in HCC1937 and MDA-MB-468 cells and lead to apoptosis. The experiment also found that compound Ilamycin E can effectively inhibit KLF5 transcription and up up the expression of the tumor suppressor factor EGR1. The comprehensive results show that the compound IlamycinE can effectively inhibit three negative breast cancer, This may provide more possibilities for the treatment of three negative breast cancer.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

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