本文選題：結(jié)腸癌 + 雷替曲塞��； 參考：《安徽醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的本研究通過體外細(xì)胞實(shí)驗(yàn)探討ERK抑制劑PD98059是否提高結(jié)腸癌細(xì)胞HT-29對(duì)RTX(雷替曲塞)的敏感性,并對(duì)其中的相關(guān)作用特點(diǎn)及機(jī)制進(jìn)行深入研究,為結(jié)腸癌的臨床治療提供可靠的實(shí)驗(yàn)數(shù)據(jù)支持。方法1、培養(yǎng)人結(jié)腸癌細(xì)胞HT-29,待培養(yǎng)至對(duì)數(shù)生長期時(shí)進(jìn)行傳代種板,細(xì)胞貼壁后按不同處理因素進(jìn)行分組:對(duì)照組、單純RTX組、單純PD98059組及聯(lián)合用藥組。2、各處理組細(xì)胞在作用24h后于普通光學(xué)倒置顯微鏡下觀察各組的細(xì)胞生長狀態(tài)及形態(tài)學(xué)改變。3、使用MTT法及細(xì)胞計(jì)數(shù)法分別檢測(cè)各實(shí)驗(yàn)組的細(xì)胞被處理24h后的細(xì)胞增殖活力。4、采用流式細(xì)胞儀法檢測(cè)被處理24h后的各實(shí)驗(yàn)組細(xì)胞的凋亡率是否出現(xiàn)差異。5、各試驗(yàn)組細(xì)胞被處理8h后,將每組的細(xì)胞總蛋白提取,使用western blot法檢測(cè)各組細(xì)胞中Ras、ERK1/2、p-ERK1/2蛋白的相對(duì)表達(dá)量。6、在無菌無酶狀態(tài)下提取出各試驗(yàn)組細(xì)胞的總RNA,反轉(zhuǎn)錄得到K-Ras、ERK1、ERK2基因的c DNA后,通過實(shí)時(shí)熒光定量PCR法檢測(cè)各組相關(guān)基因的相對(duì)表達(dá)量。結(jié)果1、倒置顯微鏡下觀察可見與對(duì)照組相比,單純PD98059組形態(tài)及數(shù)量變化不明顯,而單純RTX組與聯(lián)合用藥組細(xì)胞數(shù)量均明顯減少,并可見核碎裂、凋亡小體等典型細(xì)胞凋亡形態(tài)學(xué)變化,且聯(lián)合用藥組的細(xì)胞數(shù)量及形態(tài)學(xué)變化程度比其他各組更明顯。2、MTT法及細(xì)胞計(jì)數(shù)法結(jié)果發(fā)現(xiàn)相比于對(duì)照組,單純PD98059組細(xì)胞增殖率未出現(xiàn)明顯變化,而單純RTX組與聯(lián)合用藥組的細(xì)胞增殖活力均降低,且聯(lián)合用藥組的細(xì)胞增殖率的降低程度最深。3、流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率變化方面,可見與對(duì)照組相比各實(shí)驗(yàn)組的細(xì)胞凋亡率均明顯升高,且聯(lián)合用藥組的細(xì)胞凋亡趨勢(shì)最顯著。4、western blot法檢測(cè)細(xì)胞相關(guān)蛋白的表達(dá)量時(shí)可見與對(duì)照組相比,單純PD98059組細(xì)胞的Ras、ERK1/2蛋白表達(dá)無明顯變化,p-ERK1/2蛋白表達(dá)下調(diào);單純RTX組細(xì)胞的ERK1/2、Ras、p-ERK1/2蛋白表達(dá)無明顯差異;聯(lián)合用藥組細(xì)胞的Ras、ERK1/2蛋白表達(dá)均無明顯變化,p-ERK1/2蛋白表達(dá)均下調(diào)。5、RT-PCR結(jié)果表示各實(shí)驗(yàn)組相比于對(duì)照組在ERK1、ERK2、K-Ras mRNA的表達(dá)上均未表現(xiàn)出明顯的差異(即P0.05)。結(jié)論1、RTX對(duì)結(jié)腸癌細(xì)胞HT-29具有抑制增殖及促進(jìn)凋亡作用。2、ERK抑制劑在某種程度上可以增加RTX對(duì)HT-29的增殖抑制及凋亡促進(jìn)作用,即ERK抑制劑可增加腸癌細(xì)胞對(duì)RTX的敏感性。3、ERK抑制劑可能通過RAS-MEK-ERK通路在蛋白水平而非基因水平上增加結(jié)腸癌細(xì)胞對(duì)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(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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