葡萄籽原花青素對(duì)結(jié)腸癌細(xì)胞的抑制作用及其機(jī)制的研究
發(fā)布時(shí)間：2018-11-07 21:35
【摘要】：目的:結(jié)腸癌(colorectal cancer,CRC)的發(fā)病率和死亡率很高,研究發(fā)現(xiàn)飲食結(jié)構(gòu)與癌癥的發(fā)生密切相關(guān),其中抗氧化劑在抑制腫瘤發(fā)生過程具有重要的作用。作為重要的抗氧化物質(zhì),原花青素在人們?nèi)粘ｏ嬍辰Y(jié)構(gòu)中占有較高的比例,是藥食同源的典型代表。原花青素是一類常見的多酚類物質(zhì),研究表明這類物質(zhì)具有腫瘤防治功效,并且對(duì)人體毒副作用較低,提示其在腫瘤預(yù)防和治療中具有重要的價(jià)值。本研究利用葡萄籽原花青素(Grape Seed Proanthocyanidins,GSPs)處理結(jié)腸癌細(xì)胞株 SW480 和 SW620(這兩個(gè)細(xì)胞系來自于同一患者,其中SW480具有低轉(zhuǎn)移性特點(diǎn),而SW620具有高轉(zhuǎn)移性的特點(diǎn)),檢測(cè)其對(duì)細(xì)胞的生物學(xué)特征(細(xì)胞形態(tài)、細(xì)胞增殖、細(xì)胞周期、細(xì)胞凋亡、細(xì)胞EMT、細(xì)胞外泌體)的影響,隨后采用二代測(cè)序技術(shù),在兩種細(xì)胞株中鑒定GSPs調(diào)控的基因,差異表達(dá)基因隨后進(jìn)行KEGG Pathway富集分析,探討GSPs對(duì)結(jié)腸癌細(xì)胞SW480和SW620的作用分子機(jī)制,為結(jié)腸癌的預(yù)防治療提供新的途徑與實(shí)驗(yàn)依據(jù)。方法:1.細(xì)胞生物學(xué)特征的鑒定:(1)細(xì)胞形態(tài)觀察,采用GSPs處理SW480和SW620細(xì)胞,觀察其對(duì)細(xì)胞形態(tài)的影響;(2)細(xì)胞增殖能力檢測(cè),采用MTT法檢測(cè)GSPs對(duì)細(xì)胞存活率的影響;(3)細(xì)胞周期和凋亡檢測(cè),利用流式細(xì)胞儀檢測(cè)GSPs對(duì)兩種細(xì)胞的影響。2.分子生物學(xué)鑒定:(1)上皮細(xì)胞-間充質(zhì)轉(zhuǎn)化(epithelial to mes-enchymal transition,EMT)相關(guān)的分子的鑒定,分別采用Western blotting以及實(shí)時(shí)定量PCR(Q-PCR)方法分別檢測(cè)細(xì)胞本體以及外泌體中相關(guān)分子的表達(dá),評(píng)估GSPs對(duì)EMT事件的影響。(2)兩種細(xì)胞中GSPs調(diào)控基因的鑒定,GSPs分別處理兩種細(xì)胞,利用二代測(cè)序技術(shù)檢測(cè)細(xì)胞處理前后的轉(zhuǎn)錄組差異,通過生物信息學(xué)的分析,篩選出差異表達(dá)的基因,并對(duì)這些基因進(jìn)行GO功能以及KEGG Pathway富集分析。(3)部分靶基因的驗(yàn)證,結(jié)合數(shù)據(jù)分析的結(jié)果,利用Q-PCR和Western blotting法分別檢測(cè)GSPs處理前后信號(hào)通路相關(guān)分子的mRNA和蛋白表達(dá)水平,進(jìn)而從機(jī)制上闡明GSPs對(duì)SW480和SW620的抑制作用。結(jié)果:GSPs作用于SW480和SW620細(xì)胞后,形態(tài)均發(fā)生明顯改變,細(xì)胞發(fā)生膨脹,多呈圓形,周緣毛糙不規(guī)則。在短期時(shí)間(24h)內(nèi)GSPs對(duì)SW480和SW620細(xì)胞的增殖具有促進(jìn)作用,而在處理更長(zhǎng)時(shí)間后(48 h、72 h),則具有抑制作用,細(xì)胞的存活率顯著下降。GSPs可引起SW480和SW620細(xì)胞周期在G0/G1期發(fā)生阻滯,而且能夠顯著促進(jìn)細(xì)胞的凋亡。GSPs對(duì)SW480和SW620細(xì)胞EMT也具有抑制作用,對(duì)應(yīng)的分子變化顯示,GSPs處理后兩細(xì)胞的胞體以及外泌體里的E-cadherin的表達(dá)水平均升高,同時(shí)還發(fā)現(xiàn)外泌體中miRNA的表達(dá)量降低。差異表達(dá)基因篩選顯示,超過200個(gè)差異基因涉及到Akt的通路,進(jìn)一步對(duì)Akt通路相關(guān)的分子檢測(cè)發(fā)現(xiàn),GSPs處理不僅降低Akt的表達(dá)水平,同時(shí)也可抑制其磷酸化,同時(shí)可上調(diào)Bad、Bax、caspase 3和caspase 9 以及下調(diào) Bcl-2、CyclinD1 和 CDK4結(jié)論:本研究通過細(xì)胞學(xué)特征的觀察,發(fā)現(xiàn)GSPs對(duì)結(jié)腸癌細(xì)胞SW480和SW620的生物學(xué)特征有顯著的影響,如:抑制細(xì)胞的增殖、EMT能力,造成細(xì)胞在G0/G1期發(fā)生阻滯,促進(jìn)細(xì)胞凋亡。機(jī)制研究顯示,GSPs可導(dǎo)致兩種細(xì)胞多種基因表達(dá)的差異,其中Akt通路相關(guān)基因的改變具有一定的代表性,進(jìn)一步對(duì)該通路研究顯示,GSPs可不僅降低p-Akt的表達(dá)水平,同時(shí)也抑制Akt通路的激活。另外,GSPs可細(xì)胞增殖、EMT以及凋亡相關(guān)分子表達(dá)的顯著差異。因此,本研究為結(jié)腸癌的預(yù)防和治療提供新的途徑與實(shí)驗(yàn)依據(jù)。
[Abstract]:Objective: The incidence and mortality of colon cancer (CRC) are high, and it is found that the diet structure is closely related to the occurrence of cancer, and the antioxidant plays an important role in the inhibition of tumorigenesis. As an important antioxidant, procyanidins have a high proportion in the daily diet structure of people, and are typical representatives of the medicine and food homology. The procyanidins are a kind of common polyphenols, the study shows that this kind of substance has the function of tumor prevention and cure, and has low toxic and side effect to the human body, and it is suggested that it has important value in the prevention and treatment of tumor. The study uses the grape seed procyanidins (GSPs) to treat the colon cancer cell lines SW480 and SW620 (the two cell lines come from the same patient, where the SW480 has low metastatic characteristics, and the SW620 has the characteristics of high metastatic), and detects the biological characteristics (cell morphology, The effects of cell proliferation, cell cycle, cell apoptosis, cell EMT, extracellular matrix, and the subsequent use of second-generation sequencing technology to identify the genes regulated by GSPs in both cell lines, the differentially expressed genes were then subjected to the KEGG Pathway enrichment analysis, Objective To study the molecular mechanism of GSPs on SW480 and SW620 in colon cancer cells and to provide a new way and experimental basis for the prevention and treatment of colon cancer. Method: 1. Identification of the biological characteristics of the cells: (1) The morphology of the cells was observed, and the cells of SW480 and SW620 were treated with GSPs to observe the effect on the morphology of the cells; (2) the cell proliferation ability was detected, and the effect of GSPs on the cell survival rate was detected by the MTT method; and (3) the cell cycle and the apoptosis were detected. The effect of GSPs on the two cells was detected by flow cytometry. Molecular biological identification: (1) The identification of the related molecules of epithelial-to-mesenchymal transition (EMT). Western blotting and real-time quantitative PCR (Q-PCR) were used to detect the expression of the related molecules in the cell body and the external donor, and to assess the effect of GSPs on the EMT events. (2) the genes of the GSPs in the two cells are identified, the GSPs respectively treat the two cells, the difference of the transcription groups before and after the cell treatment is detected by using the second-generation sequencing technology, the differentially expressed genes are screened through the analysis of the bioinformatics, and the GO function and the KEGG Pathway enrichment analysis are carried out on the genes. (3) The results of partial target gene, combined with the results of data analysis, were used to detect the mRNA and protein expression level of signal path-related molecules before and after GSPs treatment by using Q-PCR and Western blotting, and then the inhibition of GSPs on SW480 and SW620 was elucidated. Results: After the GSPs acted on the SW480 and SW620 cells, the morphology of the cells changed significantly, and the cells expanded, round and irregular. The proliferation of SW480 and SW620 cells was promoted by GSPs in short-term time (24h), and the survival rate of SW480 and SW620 cells decreased significantly after the treatment for a longer period of time (48 h, 72 h). GSPs can cause the cell cycle of SW480 and SW620 to block in G0/ G1 phase, and can significantly promote cell apoptosis. GSPs also inhibited the EMT of SW480 and SW620 cells, and the corresponding molecular changes showed that the expression levels of E-cadherin in both the cell and the outer donor of the two cells were increased after GSPs treatment, while the expression of the miRNAs in the external donor was also found to be reduced. The screening of the differentially expressed genes revealed that more than 200 difference genes involved the pathway of Akt, and further detection of the Akt pathway found that the GSPs treatment not only decreased the expression level of Akt, but also inhibited its phosphorylation, and the Bad, Bax, caspase 3, and caspase 9 and the down-regulation of Bcl-2 were also upregulated. Conclusion: The results of CyclinD1 and CDK4 show that GSPs have a significant effect on the biological characteristics of SW480 and SW620 in colon cancer cells. The mechanism research shows that GSPs can cause the difference of gene expression of two kinds of cells, in which the change of Akt pathway-related gene has a certain representativeness, and further research on this pathway shows that GSPs can not only reduce the expression level of p-Akt, but also inhibit the activation of Akt pathway. In addition, the proliferation of GSPs, EMT and the expression of apoptosis-related molecules were significantly different. Therefore, this study provides a new way and experimental basis for the prevention and treatment of colon cancer.
【學(xué)位授予單位】：北京交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.35
本文編號(hào)：2317632
[Abstract]:Objective: The incidence and mortality of colon cancer (CRC) are high, and it is found that the diet structure is closely related to the occurrence of cancer, and the antioxidant plays an important role in the inhibition of tumorigenesis. As an important antioxidant, procyanidins have a high proportion in the daily diet structure of people, and are typical representatives of the medicine and food homology. The procyanidins are a kind of common polyphenols, the study shows that this kind of substance has the function of tumor prevention and cure, and has low toxic and side effect to the human body, and it is suggested that it has important value in the prevention and treatment of tumor. The study uses the grape seed procyanidins (GSPs) to treat the colon cancer cell lines SW480 and SW620 (the two cell lines come from the same patient, where the SW480 has low metastatic characteristics, and the SW620 has the characteristics of high metastatic), and detects the biological characteristics (cell morphology, The effects of cell proliferation, cell cycle, cell apoptosis, cell EMT, extracellular matrix, and the subsequent use of second-generation sequencing technology to identify the genes regulated by GSPs in both cell lines, the differentially expressed genes were then subjected to the KEGG Pathway enrichment analysis, Objective To study the molecular mechanism of GSPs on SW480 and SW620 in colon cancer cells and to provide a new way and experimental basis for the prevention and treatment of colon cancer. Method: 1. Identification of the biological characteristics of the cells: (1) The morphology of the cells was observed, and the cells of SW480 and SW620 were treated with GSPs to observe the effect on the morphology of the cells; (2) the cell proliferation ability was detected, and the effect of GSPs on the cell survival rate was detected by the MTT method; and (3) the cell cycle and the apoptosis were detected. The effect of GSPs on the two cells was detected by flow cytometry. Molecular biological identification: (1) The identification of the related molecules of epithelial-to-mesenchymal transition (EMT). Western blotting and real-time quantitative PCR (Q-PCR) were used to detect the expression of the related molecules in the cell body and the external donor, and to assess the effect of GSPs on the EMT events. (2) the genes of the GSPs in the two cells are identified, the GSPs respectively treat the two cells, the difference of the transcription groups before and after the cell treatment is detected by using the second-generation sequencing technology, the differentially expressed genes are screened through the analysis of the bioinformatics, and the GO function and the KEGG Pathway enrichment analysis are carried out on the genes. (3) The results of partial target gene, combined with the results of data analysis, were used to detect the mRNA and protein expression level of signal path-related molecules before and after GSPs treatment by using Q-PCR and Western blotting, and then the inhibition of GSPs on SW480 and SW620 was elucidated. Results: After the GSPs acted on the SW480 and SW620 cells, the morphology of the cells changed significantly, and the cells expanded, round and irregular. The proliferation of SW480 and SW620 cells was promoted by GSPs in short-term time (24h), and the survival rate of SW480 and SW620 cells decreased significantly after the treatment for a longer period of time (48 h, 72 h). GSPs can cause the cell cycle of SW480 and SW620 to block in G0/ G1 phase, and can significantly promote cell apoptosis. GSPs also inhibited the EMT of SW480 and SW620 cells, and the corresponding molecular changes showed that the expression levels of E-cadherin in both the cell and the outer donor of the two cells were increased after GSPs treatment, while the expression of the miRNAs in the external donor was also found to be reduced. The screening of the differentially expressed genes revealed that more than 200 difference genes involved the pathway of Akt, and further detection of the Akt pathway found that the GSPs treatment not only decreased the expression level of Akt, but also inhibited its phosphorylation, and the Bad, Bax, caspase 3, and caspase 9 and the down-regulation of Bcl-2 were also upregulated. Conclusion: The results of CyclinD1 and CDK4 show that GSPs have a significant effect on the biological characteristics of SW480 and SW620 in colon cancer cells. The mechanism research shows that GSPs can cause the difference of gene expression of two kinds of cells, in which the change of Akt pathway-related gene has a certain representativeness, and further research on this pathway shows that GSPs can not only reduce the expression level of p-Akt, but also inhibit the activation of Akt pathway. In addition, the proliferation of GSPs, EMT and the expression of apoptosis-related molecules were significantly different. Therefore, this study provides a new way and experimental basis for the prevention and treatment of colon cancer.
【學(xué)位授予單位】：北京交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.35
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