氯胺酮（K粉）在膀胱上皮細胞凋亡中的作用機理研究
發(fā)布時間：2018-09-14 20:30
【摘要】：目的： 通過分析用氯胺酮刺激人膀胱SV-HUC-1細胞后,誘導(dǎo)其發(fā)生細胞凋亡,為初步探討氯胺酮相關(guān)性泌尿系統(tǒng)損傷的發(fā)病機制提供理論基礎(chǔ)。 方法： 培養(yǎng)人膀胱SV-HUC-1永生化上皮細胞系,用不同濃度(Ommol/L、0.5mmol/L、1mmol/L、2mmol/L)的氯胺酮刺激SV-HUC-1細胞24小時,用Annexin V-FITC/PI雙染法流式細胞術(shù)檢測細胞凋亡率,Western免疫印跡法檢測Bax、Bcl-2、pro-caspase-3、Cleaved Caspase-3蛋白表達的相對含量,并選取上述實驗中結(jié)果最佳的氯胺酮濃度作為下一步實驗的工作濃度,繼續(xù)作用于SV-HUC-1細胞不同的時間后行上述實驗。實驗數(shù)據(jù)使用SPSS19.0統(tǒng)計軟件進行統(tǒng)計分析。 結(jié)果： 1.氯胺酮對SV-HUC-1細胞凋亡率的影響 0mmol/L、0.5mmol/L、1mmol/L、2mmol/L的氯胺酮作用SV-HUC-1細胞24小時后,流式細胞術(shù)檢測結(jié)果顯示各組細胞凋亡比率分別為(3.33±0.24)%、(5.16±0.57)%、(9.39±0.52)%、(11.33±0.40)%,各組間比較P值均0.05。相關(guān)分析結(jié)果顯示,SV-HUC-1細胞凋亡率與氯胺酮質(zhì)量濃度呈正相關(guān)(r=0.829,P0.05)。 用2mmol/L的氯胺酮分別作用SV-HUC-1細胞0h、12h、24h、48h后,流式細胞術(shù)檢測顯示各組細胞凋亡比率分別為(3.72±0.22)%、(6.45±0.32)%、(11.18±0.45)%、(12.24±0.40)%,各組間比較P值均0.05。相關(guān)分析結(jié)果顯示,SV-HUC-1細胞凋亡率與氯胺酮作用時間呈正相關(guān)(r=0.762,P0.05)。 2.氯胺酮對SV-HUC-1細胞Bax、Bcl-2、pro-caspase-3、Cleaved Caspase-3蛋白的表達的影響 O mmol/L、0.5mmol/L.1mmol/L.2mmol/L的氯胺酮作用于SV-HUC-1細胞24小時后,Bax和Cleaved Caspase-3蛋白(17KD.19KD)的表達較對照組明顯增多,Bax/Bcl-2比值增大(P0.05),且隨氯胺酮濃度的升高而表達增高(rBax=0.986,r19KD=0.888,r17KD=0.769,r Bax/Bcl-2=0.982,P值均0.05)；Bcl-2和pro-caspase-3蛋白表達較對照組逐漸下降(P0.05),且隨氯胺酮濃度的升高而表達下降(r Bcl-2=-0.963,r pro-caspase-3=-0.894,P值均0.05)。 氯胺酮(2mmol/L)作用于SV-HUC-1細胞Oh、12h、24h、48h后,Bax和Cleaved Caspase-3蛋白(17KD.19KD)的表達較對照組增多,Bax/Bcl-2比值增大(P0.05),隨作用時間的延長而表達增多(r Bax=0.951,r19KD=0.787,r17KD=0.942,r Bax/Bcl-2＝0.979,P值均0.05)；Bcl-2和pro-caspase-3蛋白表達較對照組逐漸下降(P0.05),且隨作用時間的延長而表達下降(r Bcl-2:=-0.949,r pro-caspase-3=-0.914,P值均0.05)。 結(jié)論： 氯胺酮能夠誘導(dǎo)人膀胱SV-HUC-1細胞系發(fā)生細胞凋亡,并呈濃度依賴性和時間依賴性。說明了氯胺酮可能誘導(dǎo)尿路上皮細胞發(fā)生凋亡,導(dǎo)致尿路上皮層變薄或上皮缺損,進一步對上皮下肌層組織造成損害,這可能是氯胺酮相關(guān)性泌尿系統(tǒng)損傷,在疾病發(fā)病早期的一個重要機制。
[Abstract]:Aim: to investigate the mechanism of Ketamine associated urinary system injury by inducing apoptosis of human bladder SV-HUC-1 cells stimulated by ketamine. Methods: human bladder SV-HUC-1 immortalized epithelial cell lines were cultured. SV-HUC-1 cells were stimulated with different concentrations of ketamine (Ommol/L,0.5mmol/L,1mmol/L,2mmol/L) for 24 hours. Annexin V-FITC/PI double staining flow cytometry was used to detect apoptosis rate and Western blot was used to detect the relative content of Bax,Bcl-2,pro-caspase-3,Cleaved Caspase-3 protein. The optimal concentration of ketamine was selected as the working concentration of the next step. The experiment was carried out after the SV-HUC-1 cells continued to be treated for different time. The experimental data were analyzed by SPSS19.0 statistical software. Results: 1. Effect of ketamine on apoptosis of SV-HUC-1 cells. 24 hours after Ketamine treated SV-HUC-1 cells with 1 mmol / L Ketamine, the apoptotic rates were (3.33 鹵0.24), (5.16 鹵0.57), (9.39 鹵0.52), (11.33 鹵0.40), respectively (P < 0.05). The results of correlation analysis showed that the apoptosis rate of SV-HUC-1 cells was positively correlated with the mass concentration of ketamine (P 0.05). Flow cytometry showed that the apoptotic rates of SV-HUC-1 cells in each group were (3.72 鹵0.22), (6.45 鹵0.32), (11.18 鹵0.45), (12.24 鹵0.40), respectively. The results of correlation analysis showed that the apoptosis rate of SV-HUC-1 cells was positively correlated with the time of ketamine treatment (P 0.05). Effect of ketamine on the expression of Bax,Bcl-2,pro-caspase-3,Cleaved Caspase-3 protein in SV-HUC-1 cells the expression of Cleaved Caspase-3 and Bax protein (17KD.19KD) in SV-HUC-1 cells increased significantly after treatment with ketamine O mmol/L,0.5mmol/L.1mmol/L.2mmol/L for 24 hours (P0.05), and increased with the concentration of ketamine (P0.05). The expression of rBax=0.986,r19KD=0.888,r17KD=0.769,r Bax/Bcl-2=0.982,P was higher than that of the control group (rBax=0.986,r19KD=0.888,r17KD=0.769,r Bax/Bcl-2=0.982,P = 0. 05). Compared with the control group, the expression of Bcl-2 and pro-caspase-3 protein decreased gradually (P0.05), and decreased with the increase of ketamine concentration (r Bcl-2=-0.963,r pro-caspase-3=-0.894,P). The expression of Bax and Cleaved Caspase-3 protein (17KD.19KD) in SV-HUC-1 cells treated with ketamine (2mmol/L) was higher than that in control group (P0.05), and increased with the prolongation of time (r Bax=0.951,r19KD=0.787,r17KD=0.942,r Bax/Bcl-2=0.979,P). Compared with the control group, the expression of Bcl-2 and pro-caspase-3 protein decreased gradually (P0.05), and decreased with the prolongation of the action time (r Bcl-2:=-0.949,r pro-caspase-3=-0.914,P). Conclusion: ketamine can induce apoptosis of human bladder SV-HUC-1 cell line in a concentration and time dependent manner. It is suggested that ketamine may induce apoptosis of urinary tract epithelial cells, resulting in thinning or epithelial defect of urinary tract epithelium, and further damage to the myometrium of epiglottis, which may be the injury of urinary system associated with ketamine. An important mechanism in the early stages of disease.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R694.3
本文編號：2243792
[Abstract]:Aim: to investigate the mechanism of Ketamine associated urinary system injury by inducing apoptosis of human bladder SV-HUC-1 cells stimulated by ketamine. Methods: human bladder SV-HUC-1 immortalized epithelial cell lines were cultured. SV-HUC-1 cells were stimulated with different concentrations of ketamine (Ommol/L,0.5mmol/L,1mmol/L,2mmol/L) for 24 hours. Annexin V-FITC/PI double staining flow cytometry was used to detect apoptosis rate and Western blot was used to detect the relative content of Bax,Bcl-2,pro-caspase-3,Cleaved Caspase-3 protein. The optimal concentration of ketamine was selected as the working concentration of the next step. The experiment was carried out after the SV-HUC-1 cells continued to be treated for different time. The experimental data were analyzed by SPSS19.0 statistical software. Results: 1. Effect of ketamine on apoptosis of SV-HUC-1 cells. 24 hours after Ketamine treated SV-HUC-1 cells with 1 mmol / L Ketamine, the apoptotic rates were (3.33 鹵0.24), (5.16 鹵0.57), (9.39 鹵0.52), (11.33 鹵0.40), respectively (P < 0.05). The results of correlation analysis showed that the apoptosis rate of SV-HUC-1 cells was positively correlated with the mass concentration of ketamine (P 0.05). Flow cytometry showed that the apoptotic rates of SV-HUC-1 cells in each group were (3.72 鹵0.22), (6.45 鹵0.32), (11.18 鹵0.45), (12.24 鹵0.40), respectively. The results of correlation analysis showed that the apoptosis rate of SV-HUC-1 cells was positively correlated with the time of ketamine treatment (P 0.05). Effect of ketamine on the expression of Bax,Bcl-2,pro-caspase-3,Cleaved Caspase-3 protein in SV-HUC-1 cells the expression of Cleaved Caspase-3 and Bax protein (17KD.19KD) in SV-HUC-1 cells increased significantly after treatment with ketamine O mmol/L,0.5mmol/L.1mmol/L.2mmol/L for 24 hours (P0.05), and increased with the concentration of ketamine (P0.05). The expression of rBax=0.986,r19KD=0.888,r17KD=0.769,r Bax/Bcl-2=0.982,P was higher than that of the control group (rBax=0.986,r19KD=0.888,r17KD=0.769,r Bax/Bcl-2=0.982,P = 0. 05). Compared with the control group, the expression of Bcl-2 and pro-caspase-3 protein decreased gradually (P0.05), and decreased with the increase of ketamine concentration (r Bcl-2=-0.963,r pro-caspase-3=-0.894,P). The expression of Bax and Cleaved Caspase-3 protein (17KD.19KD) in SV-HUC-1 cells treated with ketamine (2mmol/L) was higher than that in control group (P0.05), and increased with the prolongation of time (r Bax=0.951,r19KD=0.787,r17KD=0.942,r Bax/Bcl-2=0.979,P). Compared with the control group, the expression of Bcl-2 and pro-caspase-3 protein decreased gradually (P0.05), and decreased with the prolongation of the action time (r Bcl-2:=-0.949,r pro-caspase-3=-0.914,P). Conclusion: ketamine can induce apoptosis of human bladder SV-HUC-1 cell line in a concentration and time dependent manner. It is suggested that ketamine may induce apoptosis of urinary tract epithelial cells, resulting in thinning or epithelial defect of urinary tract epithelium, and further damage to the myometrium of epiglottis, which may be the injury of urinary system associated with ketamine. An important mechanism in the early stages of disease.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R694.3
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