葡萄籽原花青素对结肠癌细胞的抑制作用及其机制的研究
发布时间：2018-11-07 21:35
【摘要】：目的:结肠癌(colorectal cancer,CRC)的发病率和死亡率很高,研究发现饮食结构与癌症的发生密切相关,其中抗氧化剂在抑制肿瘤发生过程具有重要的作用。作为重要的抗氧化物质,原花青素在人们日常饮食结构中占有较高的比例,是药食同源的典型代表。原花青素是一类常见的多酚类物质,研究表明这类物质具有肿瘤防治功效,并且对人体毒副作用较低,提示其在肿瘤预防和治疗中具有重要的价值。本研究利用葡萄籽原花青素(Grape Seed Proanthocyanidins,GSPs)处理结肠癌细胞株 SW480 和 SW620(这两个细胞系来自于同一患者,其中SW480具有低转移性特点,而SW620具有高转移性的特点),检测其对细胞的生物学特征(细胞形态、细胞增殖、细胞周期、细胞凋亡、细胞EMT、细胞外泌体)的影响,随后采用二代测序技术,在两种细胞株中鉴定GSPs调控的基因,差异表达基因随后进行KEGG Pathway富集分析,探讨GSPs对结肠癌细胞SW480和SW620的作用分子机制,为结肠癌的预防治疗提供新的途径与实验依据。方法:1.细胞生物学特征的鉴定:(1)细胞形态观察,采用GSPs处理SW480和SW620细胞,观察其对细胞形态的影响;(2)细胞增殖能力检测,采用MTT法检测GSPs对细胞存活率的影响;(3)细胞周期和凋亡检测,利用流式细胞仪检测GSPs对两种细胞的影响。2.分子生物学鉴定:(1)上皮细胞-间充质转化(epithelial to mes-enchymal transition,EMT)相关的分子的鉴定,分别采用Western blotting以及实时定量PCR(Q-PCR)方法分别检测细胞本体以及外泌体中相关分子的表达,评估GSPs对EMT事件的影响。(2)两种细胞中GSPs调控基因的鉴定,GSPs分别处理两种细胞,利用二代测序技术检测细胞处理前后的转录组差异,通过生物信息学的分析,筛选出差异表达的基因,并对这些基因进行GO功能以及KEGG Pathway富集分析。(3)部分靶基因的验证,结合数据分析的结果,利用Q-PCR和Western blotting法分别检测GSPs处理前后信号通路相关分子的mRNA和蛋白表达水平,进而从机制上阐明GSPs对SW480和SW620的抑制作用。结果:GSPs作用于SW480和SW620细胞后,形态均发生明显改变,细胞发生膨胀,多呈圆形,周缘毛糙不规则。在短期时间(24h)内GSPs对SW480和SW620细胞的增殖具有促进作用,而在处理更长时间后(48 h、72 h),则具有抑制作用,细胞的存活率显著下降。GSPs可引起SW480和SW620细胞周期在G0/G1期发生阻滞,而且能够显著促进细胞的凋亡。GSPs对SW480和SW620细胞EMT也具有抑制作用,对应的分子变化显示,GSPs处理后两细胞的胞体以及外泌体里的E-cadherin的表达水平均升高,同时还发现外泌体中miRNA的表达量降低。差异表达基因筛选显示,超过200个差异基因涉及到Akt的通路,进一步对Akt通路相关的分子检测发现,GSPs处理不仅降低Akt的表达水平,同时也可抑制其磷酸化,同时可上调Bad、Bax、caspase 3和caspase 9 以及下调 Bcl-2、CyclinD1 和 CDK4结论:本研究通过细胞学特征的观察,发现GSPs对结肠癌细胞SW480和SW620的生物学特征有显著的影响,如:抑制细胞的增殖、EMT能力,造成细胞在G0/G1期发生阻滞,促进细胞凋亡。机制研究显示,GSPs可导致两种细胞多种基因表达的差异,其中Akt通路相关基因的改变具有一定的代表性,进一步对该通路研究显示,GSPs可不仅降低p-Akt的表达水平,同时也抑制Akt通路的激活。另外,GSPs可细胞增殖、EMT以及凋亡相关分子表达的显著差异。因此,本研究为结肠癌的预防和治疗提供新的途径与实验依据。
[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.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35
本文编号：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.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35
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