氯胺酮（K粉）在膀胱上皮细胞凋亡中的作用机理研究

发布时间：2018-09-14 20:30

【摘要】：目的：通过分析用氯胺酮刺激人膀胱SV-HUC-1细胞后,诱导其发生细胞凋亡,为初步探讨氯胺酮相关性泌尿系统损伤的发病机制提供理论基础。方法：培养人膀胱SV-HUC-1永生化上皮细胞系,用不同浓度(Ommol/L、0.5mmol/L、1mmol/L、2mmol/L)的氯胺酮刺激SV-HUC-1细胞24小时,用Annexin V-FITC/PI双染法流式细胞术检测细胞凋亡率,Western免疫印迹法检测Bax、Bcl-2、pro-caspase-3、Cleaved Caspase-3蛋白表达的相对含量,并选取上述实验中结果最佳的氯胺酮浓度作为下一步实验的工作浓度,继续作用于SV-HUC-1细胞不同的时间后行上述实验。实验数据使用SPSS19.0统计软件进行统计分析。结果： 1.氯胺酮对SV-HUC-1细胞凋亡率的影响 0mmol/L、0.5mmol/L、1mmol/L、2mmol/L的氯胺酮作用SV-HUC-1细胞24小时后,流式细胞术检测结果显示各组细胞凋亡比率分别为(3.33±0.24)%、(5.16±0.57)%、(9.39±0.52)%、(11.33±0.40)%,各组间比较P值均0.05。相关分析结果显示,SV-HUC-1细胞凋亡率与氯胺酮质量浓度呈正相关(r=0.829,P0.05)。用2mmol/L的氯胺酮分别作用SV-HUC-1细胞0h、12h、24h、48h后,流式细胞术检测显示各组细胞凋亡比率分别为(3.72±0.22)%、(6.45±0.32)%、(11.18±0.45)%、(12.24±0.40)%,各组间比较P值均0.05。相关分析结果显示,SV-HUC-1细胞凋亡率与氯胺酮作用时间呈正相关(r=0.762,P0.05)。 2.氯胺酮对SV-HUC-1细胞Bax、Bcl-2、pro-caspase-3、Cleaved Caspase-3蛋白的表达的影响 O mmol/L、0.5mmol/L.1mmol/L.2mmol/L的氯胺酮作用于SV-HUC-1细胞24小时后,Bax和Cleaved Caspase-3蛋白(17KD.19KD)的表达较对照组明显增多,Bax/Bcl-2比值增大(P0.05),且随氯胺酮浓度的升高而表达增高(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蛋白表达较对照组逐渐下降(P0.05),且随氯胺酮浓度的升高而表达下降(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细胞Oh、12h、24h、48h后,Bax和Cleaved Caspase-3蛋白(17KD.19KD)的表达较对照组增多,Bax/Bcl-2比值增大(P0.05),随作用时间的延长而表达增多(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蛋白表达较对照组逐渐下降(P0.05),且随作用时间的延长而表达下降(r Bcl-2:=-0.949,r pro-caspase-3=-0.914,P值均0.05)。结论：氯胺酮能够诱导人膀胱SV-HUC-1细胞系发生细胞凋亡,并呈浓度依赖性和时间依赖性。说明了氯胺酮可能诱导尿路上皮细胞发生凋亡,导致尿路上皮层变薄或上皮缺损,进一步对上皮下肌层组织造成损害,这可能是氯胺酮相关性泌尿系统损伤,在疾病发病早期的一个重要机制。
[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 卤0.24), (5.16 卤0.57), (9.39 卤0.52), (11.33 卤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 卤0.22), (6.45 卤0.32), (11.18 卤0.45), (12.24 卤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.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R694.3

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