基于miR-21介导的启膈散增强低氧下食管癌EC9706细胞顺铂敏感性机制

发布时间：2018-06-16 06:16

本文选题：EC9706细胞 + 低氧　；参考：《河南中医药大学》2016年硕士论文

【摘要】：目的探讨低氧下食管癌EC9706细胞对化疗药物顺铂的敏感性及启膈散增强低氧下食管癌EC9706细胞顺铂敏感性机制。方法1采用MTT法检测常氧和低氧下不同顺铂浓度(低浓度:0.35μg·m L-1、中浓度:0.70μg·m L-1、高浓度:1.18μg·m L-1)对食管癌EC9706细胞活性的影响;2采用流式细胞术(FCM)PI染色法检测常氧和低氧下不同浓度顺铂对细胞周期的影响;3采用流式细胞术(FCM)Annexin V/PI双染法检测常氧和低氧下不同浓度顺铂对细胞凋亡的影响;4 MTT法检测低氧下不同浓度下启膈散含药血清、顺铂单用或启膈散含药血清联合顺铂应用对EC9706细胞活性的影响;5 FCM PI染色法、Annexin V/PI双染法检测低氧下启膈散含药血清(v/v,8%)、顺铂单用(0.5μg·m L-1,1.0μg·m L-1)或启膈散含药血清(v/v,8%)联合顺铂(0.5μg·m L-1,1.0μg·m L-1)应用对食管癌EC9706细胞周期、细胞凋亡的影响;6逆转录实时荧光定量PCR(quantitative reverse transcription polymerase chain reaction,q RT-PCR)检测低氧下启膈散含药血清、顺铂单用或启膈散含药血清联合顺铂应用EC9706细胞miR-21及其下游分子programmed cell death protein 4(PDCD4)、phosphatase and tensin homologdeleted on chromosome ten(PTEN)m RNA表达量;7 Western blot法检测低氧下启膈散含药血清、顺铂单用或启膈散含药血清联合顺铂应用EC9706细胞PDCD4、PTEN蛋白表达量。结果1 MTT法检测结果显示,常氧和低氧下顺铂分别作用细胞48h后,随着顺铂浓度的增加,其吸光度(absorbance,A)逐渐降低;与常氧下同顺铂浓度相比,低氧下各组A值均高于常氧下,组间比较差异具有显著性统计学意义(P0.05);顺铂作用细胞48h后的IC50值,常氧组为1.18±0.05μM,低氧组为2.68±0.06μM,组间比较差异有显著性统计学意义(P0.05)。2流式细胞术检测细胞周期结果显示,低氧对照组较常氧对照组S期细胞增多,组间差异具有显著性统计学意义(P0.05);常氧下顺铂处理组与对照组相比较,S期细胞增多,G1期细胞减少,组间差异具有显著性统计学意义(P0.05),其中,顺铂低、中浓度组较对照组S期细胞数增加、G1期细胞减少更为显著(P0.05);低氧下顺铂处理组与对照组相比较,S期细胞数增加,G1期细胞减少,组间差异具有显著性统计学意义(P0.05);低氧与常氧下同浓度顺铂处理组比较,低氧下S期细胞明显增多,组间差异具有显著性统计学意义(P0.05)。3 Annexin V/PI双染检测细胞凋亡结果显示,常氧与低氧下顺铂处理各组分别与其对照组相比,细胞凋亡率增加,且呈剂量依赖性,组间差异具有显著性统计学意义(P0.05);低氧与常氧下同浓度顺铂处理组相比较,细胞凋亡率降低,组间差异具有显著性统计学意义(P0.05)。4启膈散含药血清在浓度为8%(含8%含药血清的RPMI 1640培养基)、10%(含10%含药血清的RPMI 1640培养基)与1.0μg·m L-1顺铂合用吸光度值(A值)显著低于启膈散含药血清组、顺铂组,其药物相互作用系数(coefficient of drug in interaction,CDI)分别为0.90、0.94。5与对照组相比,顺铂低浓度组、顺铂高浓度组、启膈散含药血清组、联合低浓度组、联合高浓度组,S期细胞数量均增多,组间差异有显著性统计学意义(P0.05)。6与对照组相比较,各组细胞凋亡率均升高,组间差异有显著性统计学意义(P0.05);联合高浓度、联合低浓度与同浓度顺铂组相比,细胞凋亡率均升高,且联合高浓度组凋亡率高于联合低浓度组,组间差异有显著性统计学意义(P0.05)。7与对照组相比较,顺铂高浓度组、联合高浓度组、联合低浓度组miR-21表达量低于对照组,组间差异有显著性统计学意义(P0.05);联合高、低浓度组miR-21表达量低于同浓度顺铂组(P0.05),且高浓度组低于低浓度组(P0.05),组间差异有显著性统计学意义。8与对照组相比较,顺铂高浓度组、启膈散含药血清组PDCD4 m RNA表达量显著低于对照组,组间差异有显著性统计学意义(P0.05);联合高、低浓度组PDCD4 m RNA、PTEN m RNA表达量高于对照组,组间差异有显著性统计学意义(P0.05);同时,联合高、低浓度组PDCD4 m RNA、PTEN m RNA显著高于同浓度顺铂组(P0.05),且联合高浓度组PDCD4 m RNA、PTEN m RNA表达高于低浓度组(P0.05),组间差异有显著性统计学意义。9与对照组相比,联合高、低浓度组PTEN蛋白表达升高(P0.05),且联合高、低浓度组PTEN蛋白明显高于同浓度单独应用顺铂组,组间差异有显著性统计学意义(P0.05);顺铂高浓度组、顺铂低浓度组、联合高浓度组、联合低浓度组PDCD4蛋白高于对照组(P0.05),联合高浓度组显著高于同浓度顺铂组和联合低浓度组(P0.05),组间差异有显著性统计学意义。结论1低氧下食管癌EC9706细胞对顺铂的敏感性较常氧下显著性降低,与低氧下EC9706细胞的活性较常氧下高,S期细胞较常氧下高,细胞凋亡率较常氧低相关。2低氧下启膈散含药血清对顺铂抑制EC9706细胞活性有协同作用。3低氧下启膈散含药血清对顺铂诱导的细胞凋亡有协同作用。4低氧下启膈散含药血清对顺铂抑制EC9706细胞活性的协同作用机制可能为抑制miR-21的表达,升高其下游因子PDCD4及PTEN的表达,从而促进食管癌EC9706细胞凋亡。
[Abstract]:Objective to investigate the sensitivity of EC9706 cells to chemotherapeutic drugs cisplatin and the mechanism of cisplatin sensitivity in hypoxic esophageal cancer EC9706 cells. Method 1 MTT was used to detect the concentration of different cisplatin (low concentration: low concentration: 0.35 mu g. M L-1, medium concentration: 0.70 u g. M L-1, high concentration: 1.18 mu g. M L-1) for esophageal cancer The effect of 6 cell activity; 2 the effect of cisplatin on the cell cycle was detected by flow cytometry (FCM) PI staining. 3 the effect of cisplatin on the cell apoptosis was detected by flow cytometry (FCM) Annexin V/PI double staining method at different concentrations of oxygen and hypoxia; and 4 MTT method was used to detect phrenic blood under different concentrations of hypoxia. The effect of cisplatin single use or phrenic powder containing serum combined with cisplatin on EC9706 cell activity; 5 FCM PI staining, Annexin V/PI double staining method for detection of phrenic powder in hypoxic serum (v/v, 8%), single use of cisplatin (0.5 mu g m L-1,1.0 micron G. M L-1) or phrenic powder (8%) combined with cisplatin EC9706 cell cycle, the effect of cell apoptosis, 6 RT real-time quantitative PCR (quantitative reverse transcription polymerase chain reaction, Q RT-PCR) detection of hypoxic phrenic drug serum, cisplatin single use or phrenic powder containing serum combined with cisplatin and downstream molecules The expression of rotein 4 (PDCD4), phosphatase and tensin homologdeleted on chromosome ten (PTEN) m RNA, serum of phrenic powder under hypoxia, serum of cisplatin single use or phrenic powder containing serum combined with cisplatin. Results 1 After 48h, the absorbance (absorbance, A) of cisplatin decreased gradually with the concentration of cisplatin, and compared with the concentration of cisplatin under normal oxygen, the A value of each group was higher than that of the normal oxygen. The difference between the groups was significant statistically significant (P0.05), the IC50 value of the fine cell 48h after cisplatin action, the 1.18 + 0.05 mu M in the normal oxygen group and 2.68 + 0 in the hypoxia group. 6 micron M, there was significant statistical difference between groups (P0.05).2 flow cytometry test cell cycle results showed that hypoxia control group compared with the normal oxygen control group S cell increase, the difference between groups had significant statistical significance (P0.05); normal oxygen cisplatin treatment group compared with the control group, S stage cell increase, G1 phase cell reduction, the difference between groups There were significant statistical significance (P0.05), among which, cisplatin was lower than that in the control group, and the number of S cells in the G1 phase was more significant than that in the control group (P0.05). Compared with the control group, the number of cells in the S phase increased and the G1 phase cells decreased, and the difference between the groups was statistically significant (P0.05), and the concentration of hypoxic and normoxic oxygen was in the same concentration. Compared with the cisplatin treatment group, the number of S cells in the hypoxic phase increased significantly. The difference between the groups had significant statistical significance (P0.05).3 Annexin V/PI double staining detection of cell apoptosis results showed that the rate of cell apoptosis increased and was dose-dependent compared with the control group, respectively, and the difference between the groups was statistically significant. Significance (P0.05); compared with cisplatin treatment group, the rate of apoptosis decreased and the difference between groups had significant statistical significance (P0.05).4 phrenic dispersive serum was 8% (RPMI 1640 medium containing 8% drug serum), 10% (containing 10% serum RPMI 1640 medium) and 1 u g. M L-1 cisplatin combined absorbance value (A value) was significantly lower than phrenic powder serum group, cisplatin group, its drug interaction coefficient (coefficient of drug in interaction, CDI) was compared with the control group, respectively, the low concentration of cisplatin group, cisplatin high concentration group, phrenic powder serum group, combined low concentration group, combined high concentration group, S stage, the number of cells increased, the difference between groups There was significant statistical significance (P0.05).6 compared with the control group, the apoptosis rate of all groups increased, there was significant difference between groups (P0.05). Combined high concentration, combined low concentration and cisplatin group, compared with the same concentration of cisplatin group, the apoptosis rate increased, and the rate of apoptosis in the combined high concentration group was higher than that of the combined low concentration group, and the difference between the groups was significant. Statistical significance (P0.05).7 compared with the control group, the expression of miR-21 in high concentration group, combined high concentration group and low concentration group was lower than that of control group, and there was significant difference between groups (P0.05). The expression of miR-21 in combined high and low concentration groups was lower than that of the same concentration group (P0.05), and the high concentration group was lower than the low concentration group (P0.05), and the difference between the groups was poor. Compared with the control group,.8 was compared with the control group. The expression of PDCD4 m RNA in cisplatin high concentration group and phrenic powder serum group was significantly lower than that of the control group, and there was significant statistical significance (P0.05). The expression of PTEN m RNA in the combined high, low concentration group, PDCD4 m RNA and PTEN m RNA were higher than those in the control group. There was significant statistical significance between the groups (P0.0). 5); at the same time, the combination of high and low concentration group PDCD4 m RNA, PTEN m RNA significantly higher than the same concentration of cisplatin group (P0.05), and the combined high concentration group PDCD4 m RNA, PTEN m is higher than the low concentration group, there is a significant difference between the groups. The PTEN protein in the degree group was significantly higher than the same concentration of cisplatin alone, and there was significant difference between the groups (P0.05). The high concentration group of cisplatin, the low concentration group of cisplatin, the combined high concentration group and the combined low concentration group were higher than the control group (P0.05), and the combined high concentration group was significantly higher than the same concentration cisplatin group and the combined low concentration group (P0.05), and the group was significantly higher than the same concentration group and the combined low concentration group (P0.05), and the group of high concentration of cisplatin was significantly higher than that of the group of the same concentration (P0.05). The difference has significant statistical significance. Conclusion the sensitivity of EC9706 cells to cisplatin in 1 hypoxic esophageal cancer is significantly lower than that under normal oxygen, and the activity of EC9706 cells under hypoxia is higher than that under normal oxygen, S phase cells are higher than normal oxygen, and the apoptosis rate is higher than that of normal oxygen low oxygen hypoxic.2 hypoxic serum containing cisplatin inhibition of EC9706 cell activity. The synergistic effect of cisplatin induced apoptosis under the same effect of.3 hypoxia was synergistic in cisplatin induced apoptosis. The synergistic mechanism of cisplatin containing serum on cisplatin inhibited EC9706 cell activity in.4 hypoxic hypoxia could inhibit the expression of miR-21, increase the expression of PDCD4 and PTEN, and promote the apoptosis of EC9706 cells in esophageal cancer.
【学位授予单位】：河南中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1

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