蒿甲醚体外对结直肠癌的放疗增敏作用及放化疗抵抗细胞株的逆转作用研究
发布时间：2019-04-23 12:53
【摘要】：[目的]大量研究发现放疗后的肿瘤细胞发生了上皮-间质转化(epithelial mesenchymal transition, EMT ),而嵩甲醚具有抗肿瘤作用,本课题拟探讨嵩甲醚对结直肠癌的放疗增敏作用及对放化疗抵抗结直肠癌的逆转作用,并探索其与上皮-转化的关系,为蒿甲醚提高结直肠癌放化疗疗效提供理论依据,为临床提高结直肠癌放化疗疗效提供实验基础。[方法]1、蒿甲醚体外对结直肠癌细胞放疗增敏作用及其与EMT关系的研究:①选取HCT116细胞株体外培养,分为对照组和蒿甲醚作用组,用MTT法检测蒿甲醚对其细胞毒作用,并计算48hIC20。用克隆形成实验对其放化疗敏感性进行鉴定,验证蒿甲醚对结直肠癌细胞株体外放疗增敏的作用。②探索蒿甲醚对结直肠癌细胞株放疗增敏的机制。选取HCT116细胞株体外培养,分为四个组:对照组(不做任何处理);蒿甲醚作用组(蒿甲醚浓度为270μg/ml);蒿甲醚+放疗组(蒿甲醚浓度为270μg/ml,放疗剂量为2Gy、6Mv);单纯同期放疗组(放疗剂量为2Gy、6Mv)。用Real-time PCR, Western blot检测E-cadherin,N-cadherin,Vimentin,SnailmRNA及蛋白,探索嵩甲醚放疗增敏作用的机制。2、结直肠癌放化疗抵抗细胞株的建立及鉴定:①选取HCT116细胞株体外培养,待细胞生长至约80%融合时将其暴露于1Oμmol/L的5-Fu中,同时在室温下给予4Gy的6MvX-ray照射,继续将细胞暴露于5-Fu培养至第24小时(从开始暴露于5-Fu中开始计算),更换新鲜培养液,待残余细胞恢复生长;②再用相同方法处理细胞11次,得到HCT116残余细胞株。用克隆形成实验鉴定残癌细胞株是否为放化疗抵抗细胞株(HCT116CRR)。3、探讨蒿甲醚逆转结直肠癌细胞的放化疗抵抗作用及其与EMT关系的研究①选取HCT116CRR细胞株体外培养,分为对照组和蒿甲醚作用组,用MTT法检测蒿甲醚对其细胞毒作用,并计算48hIC20。用克隆形成实验对其放化疗敏感性进行鉴定,验证蒿甲醚逆转放化疗抵抗结直肠癌细胞株的放化疗抵抗的作用。②探索蒿甲醚逆转放化疗抵抗结直肠癌细胞株的机制。选取HCT116CRR细胞株体外培养,分为四个组:对照组(不做任何处理);蒿甲醚作用组(蒿甲醚浓度为250μg/ml);蒿甲醚+放化疗组(蒿甲醚浓度为250μg/ml,放疗剂量为2Gy、6Mv);单纯放化疗组(放疗剂量为2Gy、6Mv)。用Real-time PCR,Western blot 检测 E-cadherin, N-cadherin, Vimentin, Snail mRNA 及蛋白,探索蒿甲醚逆转放化疗抵抗结直肠癌细胞的作用机制。[结果]1、①嵩甲醚对结直肠癌细胞株体外放疗增敏的作用。②Real-time PCR和Western blot结果显示E-cadherin mRNA及其蛋白的表达在蒿甲醚组较对照组明显升高,单纯放疗组较对照组降低,而蒿甲醚联合放疗组较蒿甲醚组降低,但比单纯放疗组升高;N-cadherin mRNA及其蛋白的表达情况,单纯放疗组较对照组升高,而蒿甲醚联合放疗组较蒿甲醚组升高,但比单纯放疗组降低;Snail、Vimentin mRNA及其蛋白的表达情况,蒿甲醚组较对照组明显降低,单纯放疗组较对照组升高,而蒿甲醚联合放疗组较蒿甲醚组升高,但比单纯放疗组降低。2、成功诱导HCT116CRR细胞株,并鉴定其为放化疗抵抗细胞株。3、①蒿甲醚逆转放化疗抵抗结直肠癌细胞株的放化疗抵抗的作用。②Real-time PCR和Western blot结果显示E-cadherin mRNA及其蛋白的表达情况,单纯放化疗组蒿甲醚联合放化疗组对照组蒿甲醚组;N-cadherin、Vimentin、Snail mRNA及其蛋白的表达情况,单纯放化疗组 嵩甲醚联合放化疗组 对照组 蒿甲醚组。[结论]1、人结直肠癌细胞经放化疗后发生了 EMT的改变。2、蒿甲醚对结直肠癌细胞株有放疗增敏作用,其作用机制与EMT相关,即上调E-cadherin mRNA及其蛋白的表达、下调N-cadherin、Vimentin、Snail mRNA及其蛋白的表达。3、蒿甲醚能逆转放化疗抵抗结直肠癌细胞株的放化疗抵抗作用,其作用机制与EMT相关,即上调E-cadherin mRNA及其蛋白的表达、下调N-cadherin、Vimentin、Snail mRNA及其蛋白的表达。
[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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
本文编号：2463486
[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.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
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