發(fā)布時間：2018-06-20 09:09

  本文選題：天然化合產(chǎn)物 + Ilamycin　； 參考：《南方醫(yī)科大學》2017年碩士論文

【摘要】：意義乳腺癌是世界范圍內(nèi)女性最為常見的惡性腫瘤,在女性因癌癥致死原因中占據(jù)主導地位。三陰性乳腺癌(TNBC)定義為缺乏雌激素受體(ER)、孕激素受體(PR)以及人表皮生長因子受體2(HER2)基因的特殊乳腺癌亞型,占乳腺癌全部類型15-20%。TNBC具有高風險的轉移與復發(fā)率,目前仍缺乏有效的治療措施。天然化合產(chǎn)物Ilamycin家族,來源于海洋微生物次級代謝產(chǎn)物,其細胞毒活性從來沒有被報道過。有初步研究結果顯示,化合物Ilamycin具有抗腫瘤活性,但仍需進一步深入研究。方法1.SRB法檢測細胞存活率,反映化合物Ilamycin E,F對乳腺癌細胞存活的影響。使用不同濃度 Ilamycin E,F 在 HCC 1806,HCC 1937,MDA-MB-468,MDA-MB-231,T47D,MCF7,SKBR3,BT474,MCF10A 細胞系驗證效果,并進一步用SRB的方法測定每株細胞系準確的IC50。最后根據(jù)實驗需要選出作為研究的代表細胞系,作為后續(xù)實驗的材料。2.EdU檢測細胞增殖為了檢測Ilamycin E對細胞增殖的影響,以不同濃度Ilamycin E(0,10,20,30μM)處理 HCC1937 和 MDA-MB-468 細胞 24 小時,以 EPI(0.2μM)作為陽性對照,然后用Click-iT EdU試劑盒進行處理,最后封片拍照,所得結果用ImageJ和IPP軟件處理,從每個處理結果中選擇3張圖片進行統(tǒng)計、處理,所得比值作為最后結果。3.細胞周期試驗為了進一步分析Ilamycin E導致細胞存活抑制的主要方式,我們使用流式細胞儀檢測化合物對細胞周期進程的影響。用IlamycinE不同濃度(0,10,20,30μM)處理HCC1937,MDA-MB-468細胞24小時,胰酶消化,75%乙醇4℃固定。最后以大約1x106個細胞用100 μl 0.6%NP40+1μl PI+1 μ1 RNase的比例配成體系,重懸細胞。染色處理,AccuriC6(BD)上機分析細胞周期,軟件FlowJo分析統(tǒng)計結果。4.細胞凋亡分析為了檢測化合物Ilamycin E對細胞凋亡的影響,我們使用Annexin V和PI雙染流式細胞方法檢測化合物Ilamycin E對細胞凋亡的影響。使用Ilamycin E不同濃度(0,10,20,30μM)處理HCC1937細胞24小時,處理MDA-MB-468細胞36小時,Annexin V和PI雙染流式細胞儀上機檢測、分析細胞凋亡情況。5.蛋白印跡檢測周期和凋亡相關蛋白的改變,用以探索凋亡機制為了探討細胞周期和凋亡改變的機制,我們做了大量蛋白篩查,使用不同濃度Ilamycin E(0,10,20,30μM)處理48小時或用30μM單濃度處理HCC1937,MDA-MB-468細胞不同時間,加細胞裂解液冰上裂解30min,離心,蛋白定量,然后加4×SDS Loading Buffer,98 ℃ 5min煮樣變性,上樣,SDS聚丙烯酰胺凝膠電泳分離蛋白,轉膜,抗體孵育14小時,二抗孵育2小時,曝光儀檢測檢測各個目標條帶變化情況。使用Photoshop作圖軟件進行數(shù)據(jù)處理。6.數(shù)據(jù)處理為了保證數(shù)據(jù)的準確性,實驗數(shù)據(jù)進行至少三次獨立重復的驗證。使用軟件SPSS運算t檢驗進行顯著性分析,P值0.05則認為有顯著性差異。流式結果使用FlowJo處理,柱形圖和線形圖使用GraphPad制作。Edu結果使用ImageJ,IPP進行重疊計數(shù)。結果1.SRB細胞存活率檢測結果顯示,化合物Ilamycin E對HCC 1806,HCC 1937,MDA-MB-468,MDA-MB-231,T47D,MCF7,SKBR3,BT474,MCF10A 細胞具有廣泛的細胞毒活性,同時化合物Ilamycin F不具有或在所給有效劑量范圍內(nèi)不具有細胞毒活性�；衔颕C50檢測結果顯示,Ilamycin E對細胞有效IC50分布為14.24-49.19μM,三陰性乳腺癌細胞HCC1937細胞系最為敏感,而人永生化乳腺上皮細胞MCF10A細胞系敏感性最差�；衔颕lamycin E與F的結果差異表明Ilamycin家族化合物細胞毒活性具有特定的化學基團,這為化合物的改造指明了方向。2.EdU細胞增殖實驗結果顯示,化合物Ilamycin E顯著抑制三陰性乳腺癌細胞系HCC1937和MDA-MB-468的DNA合成,減少細胞分裂,證明細胞增殖受到抑制。因此,我們初步得出結論,即化合物Ilamycin E導致的細胞存活抑制部分歸功于細胞增殖抑制。3.流式細胞周期檢測結果顯示,隨著化合物Ilamycin E有效作用濃度的遞增,Ilamycin E顯著增加HCC1937和MDA-MB-468兩株三陰性乳腺癌細胞G0/G1期細胞比例,并降低細胞S期和G2/M期細胞比例,化合物Ilamycin E有效抑制HCC1937 和 MDA-MB-468 細胞 G1/S 進程。4.流式細胞凋亡實驗結果顯示,化合物Ilamycin E明顯導致HCC1937和MDA-MB-468兩株三陰性乳腺癌細胞凋亡比例增加,并且隨著化合物Ilamycin E有效作用濃度的增加,細胞凋亡比例逐漸增加。實驗結果表明,化合物Ilamycin E導致的細胞存活抑制部分原因歸功于誘導細胞凋亡。5.使用蛋白印跡實驗經(jīng)過大量篩查表明,Ilamycin E明顯導致三陰性乳腺癌細胞系HCC1937和MDA-MB-468 一系列周期、凋亡相關蛋白的變化。同時,Real-time PCR檢測結果顯示,化合物Ilamycin E下調(diào)KLF5以及上調(diào)EGR1的轉錄。而ERStress信號通路相關蛋白Bip,IRE1α,XIAP,CHOP均有顯著增加。實驗結果表明,化合物IlamycinE在調(diào)控細胞周期、凋亡相關蛋白表達的同時,顯著誘導細胞ER Stress信號通路的活化。結論本研究結果顯示,Ilamycin家族成員IlamycinE具有潛在的抗三陰性乳腺癌活性�；衔颕lamycin E導致三陰性乳腺癌HCC1937和MDA-MB-468細胞周期相關蛋白Cyclin D1顯著下降,而細胞周期蛋白依賴性激酶抑制劑p21、p27顯著上升,并引起細胞G1/S期細胞周期進程阻滯�；衔颕lamycinE還能有效誘導HCC1937和MDA-MB-468細胞ER stress的產(chǎn)生,并導致細胞凋亡。實驗還發(fā)現(xiàn)化合物Ilamycin E能有效抑制KLF5轉錄,同時上調(diào)腫瘤抑制因子EGR1的表達。綜合結果表明化合物IlamycinE能有效抑制三陰性乳腺癌,這或許將為三陰性乳腺癌的治療提供更多的可能。
[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.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.9

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相關期刊論文 前1條

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