shRNA干扰沉默hARD1后对5-FU诱导大肠癌细胞凋亡的影响研究

发布时间：2018-07-15 19:22

【摘要】：目的：本实验以人大肠腺癌SW620细胞株为研究对象,在细胞水平上,探讨重组质粒hARDl-shRNA对5--Fu诱导人大肠腺癌细胞凋亡的影响,从而为靶向分子治疗、靶向分子治疗联合化疗治疗大肠癌、研究影响化疗敏感性因子提供一些实验依据,为后续hARD1的深入研究提供基础。方法：1、人大肠腺癌细胞株SW620、shCON-SW620、hARD 1-shRNA-SW620的培养。2、稳定转染细胞系pENTRTM/U6-hARDl-shRNA-SW620的转染率检测。实验分组：实验组：hARDl-shRNA稳定转染SW620细胞。阴性对照组：空载质粒转染SW620细胞。空白对照组：SW620细胞3、MTT法分别检测三个实验组对5-Fu敏感性的影响4、流式细胞术Annexin V/PI双染法检测细胞早期凋亡5、流式细胞术PI单染法检测细胞周期的变化6、Western blot检测5-Fu作用后的大肠腺癌组织中]hARDl蛋白表达7, Q-PCR检测5-Fu作用后的大肠腺癌组织中hARDl mRNA的表达结果：1、人大肠腺癌细胞株SW620、shCON-SW620、hARDl-shRNA-SW620的培养。体外培养成功且生长状态良好。2、pENTRTM/U6-hARDl-shRNA-SW620稳定转染细胞系稳定性和转染率检测：shRNA-ARDl SW620培养48h后对hARDl mRNA表达抑制率达54.8%。3、MTT法检测三个实验组对5-Fu敏感性的影响：分别用不同浓度5-Fu(0、1,、10、20、40、100、500、1000μg/ml)处理各组细胞观察各时间段(0、12、24、48、72h)细胞的生长,测吸光光度值(OD值),计算肿瘤细胞抑制率及IC50。SPSS20.0统计软件处理,单因素方差分析结果显示：瘤细胞抑制率及IC50实验组与阴性组、实验组与空白组比较有统计学意义(P0.05)；空白组与阴性对照组相比,差异无统计学意义(P0.05)。半数抑制浓度(IC50)分别为：实验组344.953±28.539μg/ml,阴性组798.383±86.59μg/ml,空白组735.338±36.530μg/ml4、流式细胞术Annexin V/PI双染法检测细胞早期凋亡：无药物处理的三组细胞凋亡率比较：实验组、阴性对照组、空白组无明显差异,差异无统计学意义(P0.05)。低浓度5-Fu(100μg/ml)处理后细胞凋亡率比较：实验组较空白组和阴性组细胞凋亡率增加,有统计学差异；高浓度5-Fu(500μg/ml)处理后,细胞凋亡率比较：实验组较空白组和阴性组细胞凋亡率增加,有统计学差异(P0.05),空白组与阴性组比较无统计学意义(P0.05)。5、流式细胞术PI单染法检测细胞周期的变化：在5-Fu无药物作用下,实验组的G0/G1期细胞比例较阴性组、空白组高(p0.05)。低浓度(100ug/m1)的作用后各组细胞的GO/G1期细胞比例增高,其中实验组G0/G1期的细胞比例较阴性组和空白组低高(p0.05)。在5-Fu高浓度(500ug/m1)作用下出现Sub-Gl峰,实验组G0/G1期细胞较无药物组下降(p0.05),但实验组与阴性对照组和空白对照组无统计学差异(p0.05),其细胞比例较低浓度及无药物作用组明显增高(p0.05)。出现Sub-Gl峰,此为亚G1峰,其出现Sub-Gl期细胞比例较无5-Fu处理和低浓度5-Fu处理时增高,实验组增高程度较阴性组和空白组高,差异具有统计学意义p0.05)。6、Western blot检测5-Fu作用后的大肠腺癌细胞中hARD1蛋白表达：对三组细胞中hARDl蛋白相对表达量进行灰度值分析,结果表明：实验组与对照组两两对比差异有统计学意义(P0.05)；空白组与阴性对照组相比,差异无统计学意义(P0.05)。7、Q-PCR检测5-Fu作用后的大肠腺癌细胞中hARDl mRNA的表达的结果表明：hARDl mRNA表达水平,实验组与阴性组、实验组与空白组比较有统计学意义(P0.05),空白组与阴性对照组相比,差异无统计学意义(P0.05)。结论：1. shRNA沉默ARD1基因能提高5-Fu在大肠腺癌SW620细胞中治疗的敏感性,具有化疗增敏作用。2. shRNA沉默hARD1基因能促进由化疗药物5-Fu引起的大肠癌SW620细胞的凋亡。3. shRNA沉默ARD1基因联合低浓度5-Fu使大肠癌SW620细胞周期阻滞在G0/G1期,联合高浓度5-Fu则促进细胞凋亡。4. shRNA沉默ARD1基因联合5-Fu可下调大肠腺癌细胞中hARDl蛋白的表达水平。5. shRNA沉默ARD1基因联合5-Fu可下调大肠腺癌细胞中hARDlmRNA表达水平
[Abstract]:Objective: To study the effect of recombinant plasmid hARDl-shRNA on the apoptosis of human colorectal adenocarcinoma cells induced by 5--Fu on the cell level of human colorectal adenocarcinoma cell line SW620, and to provide some experimental evidence for the treatment of colorectal cancer with targeted molecular therapy combined with chemotherapy and the influence of chemotherapy sensitivity factors. In order to provide the basis for further study of hARD1, methods: 1, SW620, shCON-SW620, hARD 1-shRNA-SW620 of human colorectal adenocarcinoma cell line were cultured.2, and the transfection rate of pENTRTM/U6-hARDl-shRNA-SW620 in cell line was stable. Experimental group: experimental group: hARDl-shRNA stably transfected with SW620 cells. Negative control group: empty plasmid transfected SW620 fine. Cell. Blank control group: SW620 cells 3, MTT method detected the effect of three experimental groups on 5-Fu sensitivity respectively 4, flow cytometry Annexin V/PI double staining method to detect cell early apoptosis 5, flow cytometry PI single staining method to detect cell cycle changes 6, Western blot detected the expression of]hARDl protein in colorectal adenocarcinoma after 5-Fu action 7, Q-PCR detection The expression of hARDl mRNA in colorectal adenocarcinoma after 5-Fu action: 1, the culture of SW620, shCON-SW620, hARDl-shRNA-SW620 in the human colorectal adenocarcinoma cell line. In vitro culture, successful and well grown.2, stability and transfection rate of pENTRTM/U6-hARDl-shRNA-SW620 stable transfection cell line: shRNA-ARDl SW620 culture 48h on hARDl The inhibition rate of expression was 54.8%.3. The effect of three experimental groups on the sensitivity of 5-Fu was detected by MTT method: the growth of each time segment (0,12,24,48,72h) cells were observed with different concentrations of 5-Fu (0,1, 10,20,401005001000 mu g/ml), the absorbance photometric value (o value), the tumor cell inhibition rate and the IC50.SPSS20.0 statistical software were calculated. The results of factor variance analysis showed that the tumor cell inhibition rate and the IC50 test group and the negative group were statistically significant (P0.05), and the difference was not statistically significant (P0.05) compared with the negative control group (P0.05). The median inhibitory concentration (IC50) was 344.953 + 28.539 mu g/ml in the experimental group and 798.383 + 86.59 g/ml in the negative group. The blank group was 735.338 + 36.530 g/ml4, and the flow cytometry Annexin V/PI double staining method was used to detect the early apoptosis of the cells. There was no significant difference in the apoptosis rate between the three groups without drug treatment: the experimental group and the negative control group, the difference was not statistically significant (P0.05). The comparison of the apoptosis rate after the treatment of low concentration 5-Fu (100 u g/ml): the experimental group was more empty. The apoptosis rate of cells in the white and negative groups increased statistically. After the treatment of high concentration of 5-Fu (500 u g/ml), the apoptosis rate of the cells in the experimental group was higher than that in the blank group and the negative group (P0.05). There was no significant difference between the blank group and the negative group (P0.05).5, and the flow cytometry PI single staining method was used to detect the cell cycle. The proportion of G0/G1 cells in the experimental group was higher than that in the negative group (P0.05). The proportion of GO/G1 cells in each group increased after the effect of low concentration (100ug/m1), and the proportion of the cells in the G0/G1 phase of the experimental group was higher than that of the negative group and the blank group (P0.05). The Sub-Gl of the experimental group was higher than the negative group and the blank group (P0.05). Sub-Gl was found under the action of 5-Fu high concentration (500ug/m1). Peak, G0/G1 phase cells in the experimental group were lower than that in the non drug group (P0.05), but there was no statistical difference between the experimental group and the negative control group and the blank control group (P0.05), and the proportion of the cells in the lower concentration and the non drug group increased significantly (P0.05). The Sub-Gl peak appeared, this was the sub G1 peak, and the proportion of the cells in the Sub-Gl phase was less than 5-Fu treatment and low concentration 5-Fu treatment. Shi Zenggao, the increase of the experimental group was higher than that of the negative group and the blank group, the difference was statistically significant P0.05).6, and Western blot was used to detect the expression of hARD1 protein in the colorectal adenocarcinoma cells after 5-Fu: the relative expression of hARDl protein in the three groups of cells was analyzed. The results showed that the difference between the experimental group and the control group 22 was statistically different. The difference between the blank group and the negative control group was not statistically significant (P0.05).7. The results of hARDl mRNA expression in colorectal adenocarcinoma cells after the 5-Fu action of 5-Fu showed that the mRNA expression level of hARDl, the experimental group and the negative group, the experimental group and the blank group were statistically significant (P0.05), the blank group and the negative control group were statistically significant (P0.05). The difference was not statistically significant (P0.05). Conclusion: 1. shRNA silencing ARD1 gene can improve the sensitivity of 5-Fu in SW620 cells of colorectal adenocarcinoma, with chemotherapeutic sensitization,.2. shRNA silencing hARD1 gene can promote the apoptosis of colorectal cancer SW620 cells induced by chemotherapeutic drug 5-Fu, and the low concentration of shRNA SW620 cell cycle of colon cancer is blocked in G0/G1 phase, combined with high concentration of 5-Fu,.4. shRNA silencing ARD1 gene combined 5-Fu can downregulate the expression level of hARDl protein in colorectal adenocarcinoma cells,.5. shRNA silencing ARD1 gene combined 5-Fu can down regulate the expression level of colorectal adenocarcinoma cells
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.34

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