葡萄籽原花青素對結(jié)腸癌細胞的抑制作用及其機制的研究
發(fā)布時間：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)構與癌癥的發(fā)生密切相關,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,并且對人體毒副作用較低,提示其在腫瘤預防和治療中具有重要的價值。本研究利用葡萄籽原花青素(Grape Seed Proanthocyanidins,GSPs)處理結(jié)腸癌細胞株 SW480 和 SW620(這兩個細胞系來自于同一患者,其中SW480具有低轉(zhuǎn)移性特點,而SW620具有高轉(zhuǎn)移性的特點),檢測其對細胞的生物學特征(細胞形態(tài)、細胞增殖、細胞周期、細胞凋亡、細胞EMT、細胞外泌體)的影響,隨后采用二代測序技術,在兩種細胞株中鑒定GSPs調(diào)控的基因,差異表達基因隨后進行KEGG Pathway富集分析,探討GSPs對結(jié)腸癌細胞SW480和SW620的作用分子機制,為結(jié)腸癌的預防治療提供新的途徑與實驗依據(jù)。方法:1.細胞生物學特征的鑒定:(1)細胞形態(tài)觀察,采用GSPs處理SW480和SW620細胞,觀察其對細胞形態(tài)的影響;(2)細胞增殖能力檢測,采用MTT法檢測GSPs對細胞存活率的影響;(3)細胞周期和凋亡檢測,利用流式細胞儀檢測GSPs對兩種細胞的影響。2.分子生物學鑒定:(1)上皮細胞-間充質(zhì)轉(zhuǎn)化(epithelial to mes-enchymal transition,EMT)相關的分子的鑒定,分別采用Western blotting以及實時定量PCR(Q-PCR)方法分別檢測細胞本體以及外泌體中相關分子的表達,評估GSPs對EMT事件的影響。(2)兩種細胞中GSPs調(diào)控基因的鑒定,GSPs分別處理兩種細胞,利用二代測序技術檢測細胞處理前后的轉(zhuǎn)錄組差異,通過生物信息學的分析,篩選出差異表達的基因,并對這些基因進行GO功能以及KEGG Pathway富集分析。(3)部分靶基因的驗證,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GSPs處理前后信號通路相關分子的mRNA和蛋白表達水平,進而從機制上闡明GSPs對SW480和SW620的抑制作用。結(jié)果:GSPs作用于SW480和SW620細胞后,形態(tài)均發(fā)生明顯改變,細胞發(fā)生膨脹,多呈圓形,周緣毛糙不規(guī)則。在短期時間(24h)內(nèi)GSPs對SW480和SW620細胞的增殖具有促進作用,而在處理更長時間后(48 h、72 h),則具有抑制作用,細胞的存活率顯著下降。GSPs可引起SW480和SW620細胞周期在G0/G1期發(fā)生阻滯,而且能夠顯著促進細胞的凋亡。GSPs對SW480和SW620細胞EMT也具有抑制作用,對應的分子變化顯示,GSPs處理后兩細胞的胞體以及外泌體里的E-cadherin的表達水平均升高,同時還發(fā)現(xiàn)外泌體中miRNA的表達量降低。差異表達基因篩選顯示,超過200個差異基因涉及到Akt的通路,進一步對Akt通路相關的分子檢測發(fā)現(xiàn),GSPs處理不僅降低Akt的表達水平,同時也可抑制其磷酸化,同時可上調(diào)Bad、Bax、caspase 3和caspase 9 以及下調(diào) Bcl-2、CyclinD1 和 CDK4結(jié)論:本研究通過細胞學特征的觀察,發(fā)現(xiàn)GSPs對結(jié)腸癌細胞SW480和SW620的生物學特征有顯著的影響,如:抑制細胞的增殖、EMT能力,造成細胞在G0/G1期發(fā)生阻滯,促進細胞凋亡。機制研究顯示,GSPs可導致兩種細胞多種基因表達的差異,其中Akt通路相關基因的改變具有一定的代表性,進一步對該通路研究顯示,GSPs可不僅降低p-Akt的表達水平,同時也抑制Akt通路的激活。另外,GSPs可細胞增殖、EMT以及凋亡相關分子表達的顯著差異。因此,本研究為結(jié)腸癌的預防和治療提供新的途徑與實驗依據(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.
【學位授予單位】：北京交通大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.35
本文編號：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.
【學位授予單位】：北京交通大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.35
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