氯胺酮诱导PC12细胞凋亡的内质网应激机制研究

发布时间：2018-06-06 15:47

本文选题：氯胺酮 + PC12细胞　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：氯胺酮（ketamine）作为一种非竞争性NMDA（N-methyl-D-aspartate）受体阻滞剂，可产生剂量相关的意识和感觉分离的麻醉状态，广泛用于成人和小儿麻醉。然而越来越多的研究结果显示，全身麻醉药氯胺酮可导致神经损伤，引起神经退行性疾病和长期的神经认知缺陷。因此对氯胺酮神经毒性的研究引起人们的关注。已有研究报道氯胺酮可导致啮齿类、斑马鱼、非人类灵长类神经细胞凋亡。细胞凋亡是细胞死亡的主要形式之一，有关细胞凋亡的发生机制尚未充分阐明。现在认为有三条细胞信号通路参与凋亡的发生：即线粒体通路、死亡受体通路和内质网通路。有研究报道氯胺酮可通过线粒体途径导致体外培养的人胚胎干细胞发生凋亡，但氯胺酮是否可以通过内质网应激（Endoplasmic reticulum stress, ERS）途径诱导细胞凋亡还未明确。已有研究表明，氧自由基、应激激素和细胞因子等都可以影响内质网功能，导致未/错误折叠蛋白堆积，激活内质网应激。适度的内质网应激可提高内质网处理未折叠或错误折叠蛋白的能力，维持细胞的生存；如果应激程度过强或时间过长，细胞内的稳态不能恢复，内质网应激最终将导致细胞发生凋亡。在一些神经退行性疾病如帕金森病，阿尔茨海默病和亨廷顿病等的发病机制研究中已证实内质网应激可以引起神经细胞的凋亡。葡萄糖调节蛋白78（78-kDa glucose-regulated protein，GRP78），也被称为免疫球蛋白重链结合蛋白（Immunoglobulin heavy chain binding protein，Bip），是未折叠蛋白反应的主调控者。当钙超载、氧化应激等因素导致内质网腔中未折叠蛋白聚集时，GRP78/Bip释放跨膜蛋白，引起ERS激活。因此，GRP78/Bip被视为ERS的标志性蛋白。Caspase12是caspase家族成员，它仅产生于内质网。Caspase12仅在内质网应激时被活化，是内质网应激介导凋亡途径的关键蛋白酶。基于以上研究背景，本实验中我们研究氯胺酮对PC12细胞的毒性作用，观察内质网应激在氯胺酮诱导PC12细胞损伤的过程中是否发挥了作用，为氯胺酮神经毒性机制研究提供新的思路。方法： 1MTT实验观察0.5、1、1.5、2、2.5mM氯胺酮分别作用6、12、24h对PC12细胞存活率的影响；20、30、40M Salubrinal（内质网应激抑制剂）单独或与氯胺酮共同作用对PC12细胞存活率的影响。 2Western blot方法检测内质网应激标志分子GRP78/Bip、内质网应激介导凋亡分子Caspase12蛋白的表达:1、1.5、2mM氯胺酮作用24h对PC12细胞GRP78/Bip、Caspase12蛋白的影响；1.5mM氯胺酮作用6、12、24h对PC12细胞GRP78/Bip、Caspase12蛋白的影响；加用内质网应激抑制剂Salubrinal共同作用于PC12细胞后对两种蛋白的影响。 3为了明确氯胺酮是否导致了PC12细胞的凋亡，采用流式细胞术Annexin V-PI双标染色法检测PC12细胞凋亡率，并观察内质网应激抑制剂Salubrinal对氯胺酮所致PC12细胞凋亡的影响。 4数据用均数±标准差(Mean±SD)表示，用SPSS17.0软件进行统计学分析，各组均数的比较行单因素方差分析（ANOVA），以p＜0.05为差异有统计学意义。结果： 1PC12细胞存活率的变化：与正常对照组相比，0.5mM氯胺酮作用于PC12细胞6h后细胞存活率没有明显变化（p0.05），作用12、24h后，PC12细胞存活率明显降低（p0.05）；1、1.5、2、2.5mM氯胺酮作用于PC12细胞6、12、24h后，PC12细胞存活率明显降低（p0.05），且呈浓度依赖性；20、30、40M Salubrinal单独作用于PC12细胞24h对其存活率无明显影响；30或40M Salubrinal可抑制1.5mM氯胺酮所致PC12细胞存活率的下降（p0.05）。 2内质网应激标志分子GRP78/Bip蛋白表达的变化：与正常对照组相比，1.5、2mM氯胺酮作用于PC12细胞24h后，，GRP78/Bip蛋白表达明显增高（p0.05or p0.01），呈浓度依赖性；1.5mM氯胺酮作用于PC12细胞12、24h后，GRP78/Bip蛋白表达明显增高（p0.05or p0.01），呈时间依赖性；30M Salubrinal可抑制1.5mM氯胺酮所致PC12细胞GRP78/Bip蛋白表达的增高。内质网应激介导凋亡分子Caspase12蛋白表达的变化：与正常对照组相比，1.5、2mM氯胺酮作用于PC12细胞24h后，Caspase12蛋白表达明显增高（p0.05），呈浓度依赖性；1.5mM氯胺酮作用于PC12细胞6、12、24h后，Caspase12蛋白表达明显增高（p0.05），呈时间依赖性；30M Salubrinal可抑制1.5mM氯胺酮所致PC12细胞Caspase12蛋白表达的增高。 3PC12细胞凋亡率的变化：与对照组相比，1.5mM氯胺酮作用24h后PC12细胞总凋亡率明显升高（p0.05）；30M Salubrinal可抑制1.5mM氯胺酮所致PC12细胞凋亡率的增高（p0.05）；30M Salubrinal单独作用后细胞凋亡率较对照组相比差异无统计学意义（p0.05）。结论：氯胺酮可诱导PC12细胞发生凋亡，且呈浓度依赖和时间依赖性；内质网应激介导了氯胺酮诱导的PC12细胞凋亡。
[Abstract]:Objective : ketamine ( ketamine ) , as a non - competitive NMDA ( N - methyl - D - aspartate ) receptor blocker , can produce dose - related conscious and sensory isolated anesthesia , widely used in adult and pediatric anesthesia .

It has been reported that ketamine can induce apoptosis in rodents , zebrafish and non - human primates . Apoptosis is one of the main forms of cell death . There are three cell signaling pathways involved in apoptosis : mitochondrial pathway , death receptor pathway and endoplasmic reticulum pathway .

Studies have shown that oxygen free radicals , stress hormones , cytokines and the like can affect the function of endoplasmic reticulum , lead to accumulation of unfolded protein and activate endoplasmic reticulum stress . Appropriate endoplasmic reticulum stress can improve the ability of endoplasmic reticulum to treat unfolded or misfolded protein , and maintain cell survival ;
In some neurodegenerative diseases such as Parkinson ' s disease , Alzheimer ' s disease and Huntington ' s disease , GRP78 / Bip release the transmembrane protein .

Based on the above research background , we studied the toxicity of ketamine on PC12 cells , and observed whether the endoplasmic reticulum stress played a role in the induction of PC12 cell injury induced by ketamine , and provided a new idea for the study of ketamine neurotoxicity .

Method :

The effects of 0.5 , 1 , 1.5 , 2 , 2.5 mM ketamine on the survival rate of PC12 cells were observed by MTT assay .
Effects of 20 , 30 , 40 M ulbrinal ( endoplasmic reticulum stress inhibitors ) on the survival of PC12 cells alone or in combination with ketamine .

2Western blot was used to detect the expression of GRP78 / Bip , the endoplasmic reticulum stress - mediated apoptosis molecule Caspase12 protein : 1 , 1.5 , 2 mM ketamine on the expression of GRP78 / Bip , Caspase12 protein in PC12 cells .
The effects of 1.5mM ketamine on GRP78 / Bip , Caspase12 protein in PC12 cells were studied .
The effect of the stress inhibitor of endoplasmic reticulum stress on the two proteins was studied in PC12 cells .

3 In order to clarify whether ketamine led to apoptosis of PC12 cells , the apoptosis rate of PC12 cells was detected by flow cytometry with V - PI double standard staining , and the effects of the endoplasmic reticulum stress inhibitor on apoptosis of PC12 cells induced by ketamine were observed .

The mean 卤 SD of 4 data was expressed by mean 卤 SD . Statistical analysis was performed with SPSS 17.0 software . The comparison of the mean number of each group was statistically significant ( p < 0.05 ) .

Results :

The survival rate of PC12 cells was significantly lower than that in the normal control group ( p < 0.05 ) . The survival rate of PC12 cells decreased significantly after 12 h and 24 h ( p < 0.05 ) .
1 , 1.5 , 2 , 2.5 mM ketamine , the survival rate of PC12 cells decreased significantly after 6 , 12 and 24 h of PC12 cells ( p < 0.05 ) , and the PC12 cells were dose - dependent ;
The survival rate of PC12 cells was not significantly affected by 20 , 30 , and 40 M brinal alone .
30 or 40 M . brinal inhibited the decrease in the survival rate of PC12 cells induced by 1.5 mM ketamine ( p . 05 ) .

2 . The expression of GRP78 / Bip protein in the endoplasmic reticulum stress marker molecule was significantly higher than that of the normal control group ( p0.01 or p0.01 ) , and the expression of GRP78 / Bip protein was significantly higher than that of the normal control group ( p0.05 or p0.01 ) .
After 12 h and 24 h , the expression of GRP78 / Bip protein was significantly increased ( p0.05 or p0.01 ) .
The expression of GRP78 / Bip protein in PC12 cells induced by 1 . 5mM ketamine was inhibited by 30 mg of ketamine .

Compared with the control group , the expression of Caspase - 12 protein in PC12 cells increased significantly ( p < 0.05 ) , and the expression of Caspase - 12 protein was significantly increased ( p < 0.05 ) .
After 6 , 12 and 24 h of PC12 cells , the expression of caspase12 increased significantly ( p < 0.05 ) .
The expression of Caspase12 protein in PC12 cells induced by 1.5 mM ketamine was inhibited by 30 mg of ketamine .

Compared with the control group , the apoptosis rate of PC12 cells increased significantly after 24 h ( p < 0.05 ) .
The apoptosis rate of PC12 cells induced by 1 . 5mM ketamine was inhibited by 30 mg of ketamine ( p < 0.05 ) .
Compared with the control group , the apoptosis rate was not significantly higher than that of the control group ( p < 0.05 ) .

Conclusion :

The apoptosis of PC12 cells can be induced by ketamine , and concentration - dependent and time - dependent ;
The endoplasmic reticulum stress mediated the apoptosis of PC12 cells induced by ketamine .
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R614

【引证文献】