ERK抑制剂提高结肠癌细胞对雷替曲塞敏感性及其机制的研究

发布时间：2018-06-26 18:07

本文选题：结肠癌 + 雷替曲塞　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的本研究通过体外细胞实验探讨ERK抑制剂PD98059是否提高结肠癌细胞HT-29对RTX(雷替曲塞)的敏感性,并对其中的相关作用特点及机制进行深入研究,为结肠癌的临床治疗提供可靠的实验数据支持。方法1、培养人结肠癌细胞HT-29,待培养至对数生长期时进行传代种板,细胞贴壁后按不同处理因素进行分组:对照组、单纯RTX组、单纯PD98059组及联合用药组。2、各处理组细胞在作用24h后于普通光学倒置显微镜下观察各组的细胞生长状态及形态学改变。3、使用MTT法及细胞计数法分别检测各实验组的细胞被处理24h后的细胞增殖活力。4、采用流式细胞仪法检测被处理24h后的各实验组细胞的凋亡率是否出现差异。5、各试验组细胞被处理8h后,将每组的细胞总蛋白提取,使用western blot法检测各组细胞中Ras、ERK1/2、p-ERK1/2蛋白的相对表达量。6、在无菌无酶状态下提取出各试验组细胞的总RNA,反转录得到K-Ras、ERK1、ERK2基因的c DNA后,通过实时荧光定量PCR法检测各组相关基因的相对表达量。结果1、倒置显微镜下观察可见与对照组相比,单纯PD98059组形态及数量变化不明显,而单纯RTX组与联合用药组细胞数量均明显减少,并可见核碎裂、凋亡小体等典型细胞凋亡形态学变化,且联合用药组的细胞数量及形态学变化程度比其他各组更明显。2、MTT法及细胞计数法结果发现相比于对照组,单纯PD98059组细胞增殖率未出现明显变化,而单纯RTX组与联合用药组的细胞增殖活力均降低,且联合用药组的细胞增殖率的降低程度最深。3、流式细胞仪检测细胞凋亡率变化方面,可见与对照组相比各实验组的细胞凋亡率均明显升高,且联合用药组的细胞凋亡趋势最显著。4、western blot法检测细胞相关蛋白的表达量时可见与对照组相比,单纯PD98059组细胞的Ras、ERK1/2蛋白表达无明显变化,p-ERK1/2蛋白表达下调;单纯RTX组细胞的ERK1/2、Ras、p-ERK1/2蛋白表达无明显差异;联合用药组细胞的Ras、ERK1/2蛋白表达均无明显变化,p-ERK1/2蛋白表达均下调。5、RT-PCR结果表示各实验组相比于对照组在ERK1、ERK2、K-Ras mRNA的表达上均未表现出明显的差异(即P0.05)。结论1、RTX对结肠癌细胞HT-29具有抑制增殖及促进凋亡作用。2、ERK抑制剂在某种程度上可以增加RTX对HT-29的增殖抑制及凋亡促进作用,即ERK抑制剂可增加肠癌细胞对RTX的敏感性。3、ERK抑制剂可能通过RAS-MEK-ERK通路在蛋白水平而非基因水平上增加结肠癌细胞对RTX的敏感性。
[Abstract]:Objective to investigate whether ERK inhibitor PD98059 can increase the sensitivity of human colon cancer cell line HT-29 to RTX by cell experiment in vitro, and to study the related characteristics and mechanism. To provide reliable experimental data for the clinical treatment of colon cancer. Methods 1. Human colon cancer cell line HT-29 was cultured and cultured to logarithmic growth stage. The cells were divided into two groups according to different factors: control group, RTX group. The growth state and morphological changes of cells in each treatment group were observed under ordinary optical inverted microscope after 24 hours of treatment. MTT assay and cell count method were used to detect the changes of the cells in each experimental group. The proliferation activity of the cells treated for 24 hours was determined by flow cytometry, and the apoptosis rate of the experimental groups was detected by flow cytometry, and the apoptosis rate of the experimental groups was 8h after treatment. The total protein of each group was extracted, and the relative expression of Rasract ERK1 / 2 p-ERK1 / 2 protein was detected by western blot method. The total RNAs of the cells in each group were extracted in aseptic condition, and the c-DNA of ERK1 / ERK2 gene was obtained by reverse transcription. The relative expression of related genes in each group was detected by real-time fluorescence quantitative PCR. Results 1. Compared with the control group, the morphological and quantitative changes of PD98059 group were not obvious, but the number of cells in RTX group and combination group were significantly decreased, and nuclear fragmentation could be seen. The apoptotic morphology of typical cells such as apoptotic corpuscles, and the number and morphological changes of cells in combination group were more obvious than those in other groups. The results of MTT assay and cell count method showed that compared with the control group, the number of cells in the combined treatment group was higher than that in the control group. The cell proliferation rate of PD98059 group was not significantly changed, but the cell proliferation activity of RTX group and combination group were decreased, and the degree of cell proliferation rate was the deepest in combination group. Flow cytometry was used to detect the change of cell apoptosis rate, and the cell proliferation rate of PD98059 group was significantly lower than that of PD98059 group. Compared with the control group, the apoptotic rate of each experimental group was significantly higher than that of the control group, and the apoptotic tendency of the combined treatment group was the most significant. 4western blot assay showed that compared with the control group, the expression of cell related protein was detected by Western blot assay. In PD98059 group, there was no significant change in the expression of Raschor ERK1 / 2 protein and the expression of p-ERK1 / 2 protein was down-regulated in RTX group, but there was no significant difference in ERK1 / 2 pERK1 / 2 protein expression between RTX group and PD98059 group. The results of RT-PCR showed that there was no significant difference in the expression of ERK1 / 2 mRNA between the two groups (P0.05). Conclusion (1) RTX can inhibit proliferation and promote apoptosis of human colon cancer cell line HT-29. The inhibition and apoptosis of HT-29 by RTX can be increased to some extent by the inhibitor of ERK. That is to say, ERK inhibitor can increase the sensitivity of colorectal cancer cells to RTX. 3 ERK inhibitors may increase the sensitivity of colon cancer cells to RTX at the protein level, not at the gene level, through the RAS-MEK-ERK pathway.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35

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