【摘要】：[目的]大量研究發(fā)現(xiàn)放療后的腫瘤細(xì)胞發(fā)生了上皮-間質(zhì)轉(zhuǎn)化(epithelial mesenchymal transition, EMT ),而嵩甲醚具有抗腫瘤作用,本課題擬探討嵩甲醚對(duì)結(jié)直腸癌的放療增敏作用及對(duì)放化療抵抗結(jié)直腸癌的逆轉(zhuǎn)作用,并探索其與上皮-轉(zhuǎn)化的關(guān)系,為蒿甲醚提高結(jié)直腸癌放化療療效提供理論依據(jù),為臨床提高結(jié)直腸癌放化療療效提供實(shí)驗(yàn)基礎(chǔ)。[方法]1、蒿甲醚體外對(duì)結(jié)直腸癌細(xì)胞放療增敏作用及其與EMT關(guān)系的研究:①選取HCT116細(xì)胞株體外培養(yǎng),分為對(duì)照組和蒿甲醚作用組,用MTT法檢測(cè)蒿甲醚對(duì)其細(xì)胞毒作用,并計(jì)算48hIC20。用克隆形成實(shí)驗(yàn)對(duì)其放化療敏感性進(jìn)行鑒定,驗(yàn)證蒿甲醚對(duì)結(jié)直腸癌細(xì)胞株體外放療增敏的作用。②探索蒿甲醚對(duì)結(jié)直腸癌細(xì)胞株放療增敏的機(jī)制。選取HCT116細(xì)胞株體外培養(yǎng),分為四個(gè)組:對(duì)照組(不做任何處理);蒿甲醚作用組(蒿甲醚濃度為270μg/ml);蒿甲醚+放療組(蒿甲醚濃度為270μg/ml,放療劑量為2Gy、6Mv);單純同期放療組(放療劑量為2Gy、6Mv)。用Real-time PCR, Western blot檢測(cè)E-cadherin,N-cadherin,Vimentin,SnailmRNA及蛋白,探索嵩甲醚放療增敏作用的機(jī)制。2、結(jié)直腸癌放化療抵抗細(xì)胞株的建立及鑒定:①選取HCT116細(xì)胞株體外培養(yǎng),待細(xì)胞生長(zhǎng)至約80%融合時(shí)將其暴露于1Oμmol/L的5-Fu中,同時(shí)在室溫下給予4Gy的6MvX-ray照射,繼續(xù)將細(xì)胞暴露于5-Fu培養(yǎng)至第24小時(shí)(從開始暴露于5-Fu中開始計(jì)算),更換新鮮培養(yǎng)液,待殘余細(xì)胞恢復(fù)生長(zhǎng);②再用相同方法處理細(xì)胞11次,得到HCT116殘余細(xì)胞株。用克隆形成實(shí)驗(yàn)鑒定殘癌細(xì)胞株是否為放化療抵抗細(xì)胞株(HCT116CRR)。3、探討蒿甲醚逆轉(zhuǎn)結(jié)直腸癌細(xì)胞的放化療抵抗作用及其與EMT關(guān)系的研究①選取HCT116CRR細(xì)胞株體外培養(yǎng),分為對(duì)照組和蒿甲醚作用組,用MTT法檢測(cè)蒿甲醚對(duì)其細(xì)胞毒作用,并計(jì)算48hIC20。用克隆形成實(shí)驗(yàn)對(duì)其放化療敏感性進(jìn)行鑒定,驗(yàn)證蒿甲醚逆轉(zhuǎn)放化療抵抗結(jié)直腸癌細(xì)胞株的放化療抵抗的作用。②探索蒿甲醚逆轉(zhuǎn)放化療抵抗結(jié)直腸癌細(xì)胞株的機(jī)制。選取HCT116CRR細(xì)胞株體外培養(yǎng),分為四個(gè)組:對(duì)照組(不做任何處理);蒿甲醚作用組(蒿甲醚濃度為250μg/ml);蒿甲醚+放化療組(蒿甲醚濃度為250μg/ml,放療劑量為2Gy、6Mv);單純放化療組(放療劑量為2Gy、6Mv)。用Real-time PCR,Western blot 檢測(cè) E-cadherin, N-cadherin, Vimentin, Snail mRNA 及蛋白,探索蒿甲醚逆轉(zhuǎn)放化療抵抗結(jié)直腸癌細(xì)胞的作用機(jī)制。[結(jié)果]1、①嵩甲醚對(duì)結(jié)直腸癌細(xì)胞株體外放療增敏的作用。②Real-time PCR和Western blot結(jié)果顯示E-cadherin mRNA及其蛋白的表達(dá)在蒿甲醚組較對(duì)照組明顯升高,單純放療組較對(duì)照組降低,而蒿甲醚聯(lián)合放療組較蒿甲醚組降低,但比單純放療組升高;N-cadherin mRNA及其蛋白的表達(dá)情況,單純放療組較對(duì)照組升高,而蒿甲醚聯(lián)合放療組較蒿甲醚組升高,但比單純放療組降低;Snail、Vimentin mRNA及其蛋白的表達(dá)情況,蒿甲醚組較對(duì)照組明顯降低,單純放療組較對(duì)照組升高,而蒿甲醚聯(lián)合放療組較蒿甲醚組升高,但比單純放療組降低。2、成功誘導(dǎo)HCT116CRR細(xì)胞株,并鑒定其為放化療抵抗細(xì)胞株。3、①蒿甲醚逆轉(zhuǎn)放化療抵抗結(jié)直腸癌細(xì)胞株的放化療抵抗的作用。②Real-time PCR和Western blot結(jié)果顯示E-cadherin mRNA及其蛋白的表達(dá)情況,單純放化療組蒿甲醚聯(lián)合放化療組對(duì)照組蒿甲醚組;N-cadherin、Vimentin、Snail mRNA及其蛋白的表達(dá)情況,單純放化療組 嵩甲醚聯(lián)合放化療組 對(duì)照組 蒿甲醚組。[結(jié)論]1、人結(jié)直腸癌細(xì)胞經(jīng)放化療后發(fā)生了 EMT的改變。2、蒿甲醚對(duì)結(jié)直腸癌細(xì)胞株有放療增敏作用,其作用機(jī)制與EMT相關(guān),即上調(diào)E-cadherin mRNA及其蛋白的表達(dá)、下調(diào)N-cadherin、Vimentin、Snail mRNA及其蛋白的表達(dá)。3、蒿甲醚能逆轉(zhuǎn)放化療抵抗結(jié)直腸癌細(xì)胞株的放化療抵抗作用,其作用機(jī)制與EMT相關(guān),即上調(diào)E-cadherin mRNA及其蛋白的表達(dá)、下調(diào)N-cadherin、Vimentin、Snail mRNA及其蛋白的表達(dá)。
[Abstract]:[Objective] To study the effect of mether on the radiosensitization of colorectal cancer and the reversal of radiotherapy and chemotherapy in the treatment of colorectal cancer. And to provide a theoretical basis for improving the curative effect of the radiotherapy and chemotherapy of the colorectal cancer by the artemether, and provides an experimental basis for clinical improvement of the curative effect of the radiotherapy and chemotherapy of the colorectal cancer. [Methods] 1. The effect of artemether in vitro on the radiosensitization of colorectal cancer cells and its relationship with EMT: the in vitro culture of HCT116 cell line was selected and divided into control group and artemether group, and the cytotoxic effect of artemether was detected by MTT method, and 48 hIC20 was calculated. The effect of artemether on the radiosensitization of colorectal cancer cell line in vitro was verified by the identification of the radiochemotherapy sensitivity. The mechanism of the sensitivity of artemether to the radiotherapy of colorectal cancer cell line. The HCT116 cell line was selected to be cultured in vitro, divided into four groups: control group (no treatment), artemether group (artemether concentration of 270 & mu; g/ ml), artemether + radiotherapy group (artemether concentration of 270 & mu; g/ ml, radiotherapy dose of 2Gy, 6Mv), and simple concurrent radiotherapy group (radiation dose of 2 Gy, 6Mv). E-cadherin, N-cadherin, Vimentin, Sailan mRNA and protein were detected by Real-time PCR and Western blot. At the same time,4 Gy of 6MvX-ray irradiation was given at room temperature, the cells were continued to be exposed to 5-Fu for 24 hours (starting from the start of exposure to 5-Fu), the fresh culture solution was replaced, and the residual cells were restored to growth, and the cells were treated with the same method for 11 times to obtain the HCT116 residual cell line. The effect of artemether on the chemoradiotherapy resistance of colorectal cancer cells and its relationship with EMT were studied by means of the clone formation experiment. In vitro culture of the HCT116CRR cell line was selected and divided into the control group and the artemether action group. The cytotoxicity of artemether was detected by MTT method, and 48 hIC20 was calculated. The sensitivity of chemoradiotherapy was identified by the clone formation experiment, and the effect of artemether on the chemoradiotherapy resistance of the colorectal cancer cell line was verified. Objective To explore the mechanism of reverse chemoradiotherapy of artemether in resisting colorectal cancer cell line. The HCT116CRR cell line was selected to be cultured in vitro, divided into four groups: control group (no treatment), artemether group (artemether concentration of 250. mu.g/ ml), artemether + chemoradiotherapy group (artemether concentration of 250 & mu; g/ ml, radiotherapy dose of 2Gy, 6Mv), and radiotherapy and chemotherapy group (2 Gy for radiotherapy). 6Mv). E-cadherin, N-cadherin, Vimentin, Snail mRNA and protein were detected by Real-time PCR and Western blot. [Results] 1. The effect of methyl-methyl-methyl ether on the in vitro radiosensitization of colorectal cancer cell line. The results of Real-time PCR and Western blot showed that the expression of E-cadherin mRNA and its protein was significantly higher in the control group than in the control group. The expression of Snail, Vimentin mRNA and its protein was significantly lower in the group of artemether group than that in the control group, and the expression of Snail, Vimentin mRNA and its protein was significantly lower than that in the control group. While the combination of artemether was higher than that of the group of artemether, but it was lower than that of the simple radiotherapy group.2, the HCT116CRR cell line was successfully induced, and it was identified as the chemoradiotherapy-resistant cell line. The expression of E-cadherin mRNA and its protein was shown by Real-time PCR and Western blot. [Conclusion] 1. The change of EMT after chemoradiotherapy of human colorectal cancer cells.2. The effect of artemether on the radiosensitization of colorectal cancer cell line is related to EMT, that is, the expression of E-cadherin mRNA and its protein is up-regulated, and the expression of N-cadherin, Vimentin, Snail mRNA and its protein is down-regulated. The effect of artemether on the chemoradiotherapy resistance of the colorectal cancer cell line can be reversed by chemoradiotherapy, the mechanism of action is related to EMT, that is, the expression of E-cadherin mRNA and its protein is up-regulated, and the expression of N-cadherin, Vimentin, Snail mRNA and its protein is down-regulated.
【學(xué)位授予單位】：昆明醫(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)】：R735.34

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