發(fā)布時間：2018-06-16 06:16

  本文選題：EC9706細(xì)胞 + 低氧�。� 參考：《河南中醫(yī)藥大學(xué)》2016年碩士論文

【摘要】：目的探討低氧下食管癌EC9706細(xì)胞對化療藥物順鉑的敏感性及啟膈散增強(qiáng)低氧下食管癌EC9706細(xì)胞順鉑敏感性機(jī)制。方法1采用MTT法檢測常氧和低氧下不同順鉑濃度(低濃度:0.35μg·m L-1、中濃度:0.70μg·m L-1、高濃度:1.18μg·m L-1)對食管癌EC9706細(xì)胞活性的影響;2采用流式細(xì)胞術(shù)(FCM)PI染色法檢測常氧和低氧下不同濃度順鉑對細(xì)胞周期的影響;3采用流式細(xì)胞術(shù)(FCM)Annexin V/PI雙染法檢測常氧和低氧下不同濃度順鉑對細(xì)胞凋亡的影響;4 MTT法檢測低氧下不同濃度下啟膈散含藥血清、順鉑單用或啟膈散含藥血清聯(lián)合順鉑應(yīng)用對EC9706細(xì)胞活性的影響;5 FCM PI染色法、Annexin V/PI雙染法檢測低氧下啟膈散含藥血清(v/v,8%)、順鉑單用(0.5μg·m L-1,1.0μg·m L-1)或啟膈散含藥血清(v/v,8%)聯(lián)合順鉑(0.5μg·m L-1,1.0μg·m L-1)應(yīng)用對食管癌EC9706細(xì)胞周期、細(xì)胞凋亡的影響;6逆轉(zhuǎn)錄實時熒光定量PCR(quantitative reverse transcription polymerase chain reaction,q RT-PCR)檢測低氧下啟膈散含藥血清、順鉑單用或啟膈散含藥血清聯(lián)合順鉑應(yīng)用EC9706細(xì)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表達(dá)量;7 Western blot法檢測低氧下啟膈散含藥血清、順鉑單用或啟膈散含藥血清聯(lián)合順鉑應(yīng)用EC9706細(xì)胞PDCD4、PTEN蛋白表達(dá)量。結(jié)果1 MTT法檢測結(jié)果顯示,常氧和低氧下順鉑分別作用細(xì)胞48h后,隨著順鉑濃度的增加,其吸光度(absorbance,A)逐漸降低;與常氧下同順鉑濃度相比,低氧下各組A值均高于常氧下,組間比較差異具有顯著性統(tǒng)計學(xué)意義(P0.05);順鉑作用細(xì)胞48h后的IC50值,常氧組為1.18±0.05μM,低氧組為2.68±0.06μM,組間比較差異有顯著性統(tǒng)計學(xué)意義(P0.05)。2流式細(xì)胞術(shù)檢測細(xì)胞周期結(jié)果顯示,低氧對照組較常氧對照組S期細(xì)胞增多,組間差異具有顯著性統(tǒng)計學(xué)意義(P0.05);常氧下順鉑處理組與對照組相比較,S期細(xì)胞增多,G1期細(xì)胞減少,組間差異具有顯著性統(tǒng)計學(xué)意義(P0.05),其中,順鉑低、中濃度組較對照組S期細(xì)胞數(shù)增加、G1期細(xì)胞減少更為顯著(P0.05);低氧下順鉑處理組與對照組相比較,S期細(xì)胞數(shù)增加,G1期細(xì)胞減少,組間差異具有顯著性統(tǒng)計學(xué)意義(P0.05);低氧與常氧下同濃度順鉑處理組比較,低氧下S期細(xì)胞明顯增多,組間差異具有顯著性統(tǒng)計學(xué)意義(P0.05)。3 Annexin V/PI雙染檢測細(xì)胞凋亡結(jié)果顯示,常氧與低氧下順鉑處理各組分別與其對照組相比,細(xì)胞凋亡率增加,且呈劑量依賴性,組間差異具有顯著性統(tǒng)計學(xué)意義(P0.05);低氧與常氧下同濃度順鉑處理組相比較,細(xì)胞凋亡率降低,組間差異具有顯著性統(tǒng)計學(xué)意義(P0.05)。4啟膈散含藥血清在濃度為8%(含8%含藥血清的RPMI 1640培養(yǎng)基)、10%(含10%含藥血清的RPMI 1640培養(yǎng)基)與1.0μg·m L-1順鉑合用吸光度值(A值)顯著低于啟膈散含藥血清組、順鉑組,其藥物相互作用系數(shù)(coefficient of drug in interaction,CDI)分別為0.90、0.94。5與對照組相比,順鉑低濃度組、順鉑高濃度組、啟膈散含藥血清組、聯(lián)合低濃度組、聯(lián)合高濃度組,S期細(xì)胞數(shù)量均增多,組間差異有顯著性統(tǒng)計學(xué)意義(P0.05)。6與對照組相比較,各組細(xì)胞凋亡率均升高,組間差異有顯著性統(tǒng)計學(xué)意義(P0.05);聯(lián)合高濃度、聯(lián)合低濃度與同濃度順鉑組相比,細(xì)胞凋亡率均升高,且聯(lián)合高濃度組凋亡率高于聯(lián)合低濃度組,組間差異有顯著性統(tǒng)計學(xué)意義(P0.05)。7與對照組相比較,順鉑高濃度組、聯(lián)合高濃度組、聯(lián)合低濃度組miR-21表達(dá)量低于對照組,組間差異有顯著性統(tǒng)計學(xué)意義(P0.05);聯(lián)合高、低濃度組miR-21表達(dá)量低于同濃度順鉑組(P0.05),且高濃度組低于低濃度組(P0.05),組間差異有顯著性統(tǒng)計學(xué)意義。8與對照組相比較,順鉑高濃度組、啟膈散含藥血清組PDCD4 m RNA表達(dá)量顯著低于對照組,組間差異有顯著性統(tǒng)計學(xué)意義(P0.05);聯(lián)合高、低濃度組PDCD4 m RNA、PTEN m RNA表達(dá)量高于對照組,組間差異有顯著性統(tǒng)計學(xué)意義(P0.05);同時,聯(lián)合高、低濃度組PDCD4 m RNA、PTEN m RNA顯著高于同濃度順鉑組(P0.05),且聯(lián)合高濃度組PDCD4 m RNA、PTEN m RNA表達(dá)高于低濃度組(P0.05),組間差異有顯著性統(tǒng)計學(xué)意義。9與對照組相比,聯(lián)合高、低濃度組PTEN蛋白表達(dá)升高(P0.05),且聯(lián)合高、低濃度組PTEN蛋白明顯高于同濃度單獨(dú)應(yīng)用順鉑組,組間差異有顯著性統(tǒng)計學(xué)意義(P0.05);順鉑高濃度組、順鉑低濃度組、聯(lián)合高濃度組、聯(lián)合低濃度組PDCD4蛋白高于對照組(P0.05),聯(lián)合高濃度組顯著高于同濃度順鉑組和聯(lián)合低濃度組(P0.05),組間差異有顯著性統(tǒng)計學(xué)意義。結(jié)論1低氧下食管癌EC9706細(xì)胞對順鉑的敏感性較常氧下顯著性降低,與低氧下EC9706細(xì)胞的活性較常氧下高,S期細(xì)胞較常氧下高,細(xì)胞凋亡率較常氧低相關(guān)。2低氧下啟膈散含藥血清對順鉑抑制EC9706細(xì)胞活性有協(xié)同作用。3低氧下啟膈散含藥血清對順鉑誘導(dǎo)的細(xì)胞凋亡有協(xié)同作用。4低氧下啟膈散含藥血清對順鉑抑制EC9706細(xì)胞活性的協(xié)同作用機(jī)制可能為抑制miR-21的表達(dá),升高其下游因子PDCD4及PTEN的表達(dá),從而促進(jìn)食管癌EC9706細(xì)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.
【學(xué)位授予單位】：河南中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.1

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