过表达Derlin-1对香烟烟雾提取物诱导Ⅱ型肺泡上皮细胞凋亡的作用研究

发布时间：2018-12-14 19:16

【摘要】：目的:观察过表达Derlin-1的RLE-6TN细胞经香烟烟雾提取物(cigarette smoke extrac,CSE)不同时间处理后的凋亡率的变化。探索其与内质网应激诱导性凋亡(Endoplasmic reticulum stress induced apoptosis,ERSIA)的关系。方法:携带Derlin-1编码序列的慢病毒转染RLE-6TN细胞过表达Derlin-1基因;转染空白慢病毒作为对照组;制备CSE;用10%CSE处理对照组、过表达组细胞0h、3h、6h、12h、24h;流式细胞仪检测各组细胞的凋亡率;Western blot技术检测各组细胞Derlin-1、IRE1、p-IRE1、JNK、P-JNK,CHOP、caspase12蛋白的表达;Realtime-PCR技术检测各组细胞Derlin-1m RNA的表达。结果:1.慢病毒过表达Derlin-1稳定株的检测:Western blot检测过表达组Derlin-1蛋白的表达较对照组增加,差异有统计学意义(p0.05)。Realtime-PCR检测Derlin-1m RNA的表达结果与Western blot结果一致。2.流式细胞技术检测细胞凋亡:用10%CSE处理对照组、过表达组细胞0h,3h,6h,12h,24h后收集细胞,用Annexin V-APC/7-AAD细胞凋亡检测试剂盒处理后在流式细胞仪上检测细胞凋亡率。对照组及过表达组凋亡率随着处理时间延长而增加,每组各时间点之间两两比较差异均有统计学意义(p0.05)。过表达组凋亡率较对照组均明显降低,同一时间点的两组比较差异均有统计学意义(p0.05)。3.Derlin-1在各组细胞中的表达:Western blot结果显示对照组Derlin-1的表达随CSE处理时间的延长呈逐渐增加的趋势,且组与组之间差异均有统计学意义(p0.05)。在过表达组,Derlin-1的表达同样呈现出随时间延长的增长趋势,各组间的两两比较均有统计学差异(p0.05)。但每组较对照组表达量明显升高,差异具有统计学意义(p0.05)。Realtime-PCR检测两组Derlin-1m RNA的表达结果与Western blot结果一致。4.Derlin-1对ERS通路的影响:Western blot结果显示在对照组及过表达组中总的IRE1的表达在各时间点的变化不大,差异没有统计学意义(p0.05)。两组的p-IRE1均有随处理时间的延长呈现增加趋势,每组各时间点的比较具有统计学意义(p0.05)。但过表达组的p-IRE1的表达量较对照组有所下降,差异具有统计学意义(p0.05)5.Derlin-1对ERSIA通路的影响:Western blot结果显示在对照组和过表达组中总JNK的表达量在各时间点的表达无明显变化,差异无统计学意义(p0.05)。p-JNK与CHOP的表达量在两组中均有逐渐增加的趋势,呈时间依赖性,每组各时间点两两比较差异具有统计学意义(p0.05)。两种蛋白过表达组较对照组表达量均下降,差异具有统计学意义(p0.05)。caspase12的表达量在对照组从3h升高,12h达高峰,24h有所下降,但仍高于0h组,各时间点的两两比较差异有统计学意义(p0.05)。在过表达组中,caspase12的表达随时间的延长而增加,但各时间点表达量较对照组均下降,且差异有统计学意义(p0.05)。结论:CSE诱导RLE-6TN细胞发生ERS,促进ERSIA的发生。Derlin-1的上调可以缓解ERS,保护细胞免于凋亡。
[Abstract]:Aim: to observe the changes of apoptosis rate of RLE-6TN cells which expressed Derlin-1 after treated with cigarette smoke extract (cigarette smoke extrac,CSE) at different time. To explore the relationship between endoplasmic reticulum stress-induced apoptosis (Endoplasmic reticulum stress induced apoptosis,ERSIA). Methods: lentivirus carrying Derlin-1 coding sequence was transfected into RLE-6TN cells to overexpression Derlin-1 gene, blank lentivirus was transfected as control group, and CSE; was treated with 10%CSE for 24 h. Flow cytometry was used to detect the apoptosis rate of cells in each group. The expression of Derlin-1,IRE1,p-IRE1,JNK,P-JNK,CHOP,caspase12 protein was detected by; Western blot and the expression of Derlin-1m RNA was detected by Realtime-PCR technique. Results: 1. Detection of lentivirus overexpression Derlin-1 stable strain the expression of Derlin-1 protein in: Western blot overexpression group was significantly higher than that in control group (p0.05). The expression of Derlin-1m RNA by Realtime-PCR was consistent with that of Western blot. 2. Flow cytometry was used to detect apoptosis: the control group was treated with 10%CSE, and the cells in the overexpression group were collected after 24 hours after 0 h, 3 h, 6 h and 12 h. Apoptosis rate was detected by flow cytometry after treated with Annexin V-APC/7-AAD cell apoptosis detection kit. The apoptotic rate of the control group and the overexpression group increased with the prolongation of the treatment time, and there were significant differences between the two groups at each time point (p0.05). The rate of apoptosis in overexpression group was significantly lower than that in control group. There were significant differences between the two groups at the same time point (p0.05). The expression of: Western blot in the cells of each group showed that the expression of Derlin-1 in the control group increased gradually with the prolongation of the time of CSE treatment. The difference between the two groups was statistically significant (p 0.05). In the overexpression group, the expression of Derlin-1 also showed an increasing trend with time, and there was statistical difference between the two groups (p0. 05). However, the expression of protein in each group was significantly higher than that in the control group. The difference was statistically significant (p0.05). The expression of Derlin-1m RNA in the two groups by Realtime-PCR was consistent with that of Western blot. The effect of 4.Derlin-1 on the ERS pathway was found in the control group and the overexpression group. The expression of IRE1 changed little at different time points. The difference was not statistically significant (p0.05). The p-IRE1 of both groups showed an increasing trend with the prolongation of treatment time, and the comparison of each time point in each group was statistically significant (p0. 05). However, the expression of p-IRE1 in the overexpression group was lower than that in the control group. The difference was statistically significant (p0.05) the effect of 5.Derlin-1 on the ERSIA pathway. The results of: Western blot showed that the expression of total JNK in control group and overexpression group had no significant change at each time point. There was no significant difference (p0.05). The expression of p-JNK and CHOP increased gradually in both groups in a time-dependent manner, and the differences were statistically significant at each time point (p0.05). The expression of caspase12 increased from 3 h to 12 h, peaked at 12 h, decreased at 24 h, but still higher than that in 0 h group. There was significant difference between the two groups at each time point (p 0.05). In the overexpression group, the expression of caspase12 increased with the prolongation of time, but the expression of caspase12 at each time point was lower than that of the control group, and the difference was statistically significant (p0.05). Conclusion: CSE induces the occurrence of ERS, in RLE-6TN cells, and the up-regulation of Derlin-1 can alleviate the apoptosis of ERS,.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R56

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