甘氨酸受体细胞保护作用的机制研究

发布时间：2018-03-17 13:28

本文选题：甘氨酸受体　切入点：ATP耗竭　出处：《南京医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的：明确甘氨酸受体(Glycine receptor，GlyR)介导甘氨酸细胞保护作用的下游信号通路，阐明甘氨酸拮抗细胞ATP耗竭性损伤的分子机制，为发现新型细胞保护剂提供理论依据。方法：本研究首先使用呼吸链抑制剂联合无糖培养基共同作用复制细胞ATP耗竭模型。将犬肾细胞(Madin-Darby canine kidney，MDCK)分为ATP耗竭组和Gly(Glycine)保护组，运用Western blot检测ERK1／2、p38MAPK、JNK1／2和AKT的磷酸化活性变化。分别运用ERK1／2和AKT的阻断剂PD98059、LY294002，观察各组LDH释放率的变化。将构建好的pcDNA3.1-GlyRal真核表达载体，应用脂质体介导的转染技术将该质粒导入野生型人胚肾细胞(Humanembryonic kidney 293，HEK293)，再用G418筛选稳定的细胞表达系。将稳定表达细胞系和野生型HEK293细胞处于ATP耗竭和Gly保护状态下，运用Western blot检测ERK1／2、p38MAPK、JNK1／2和AKT的磷酸化活性变化。选用使MDCK细胞中GlyRal表达下调最明显的siRNA2，脂质体法转染入MDCK细胞，于转染后48小时使其处于ATP耗竭状态，运用Western blot观察siRNA2对GlyRα1表达的抑制效果，并运用Western blot检测ERK1／2、p38MAPK、JNK1／2和AKT的磷酸化活性变化。结果： MDCK细胞处于ATP耗竭状态下15min、30min、1h、2h后，Gly保护组与ATP耗竭组相比，ERK1／2、AKT的磷酸化活性均增强，p38MAPK的磷酸化活性减弱，而JNK1／2的磷酸化活性没有变化。分别加入阻断剂PD98059和LY294002以后，Gly保护组的LDH释放率与对照组相比均有升高。将稳定表达GlyRα1的HEK293细胞和野生型HEK293细胞处于ATP耗竭状态1h。稳定表达细胞系中，Gly保护组与ATP耗竭组相比，ERK1／2、AKT的磷酸化活性均增强，p38MAPK的磷酸化活性减弱，JNK1／2的磷酸化活性没有改变。野生型HEK293细胞中，Gly保护组和ATP耗竭组相比，ERK1／2、p38MAPK、JNK1／2和AKT的磷酸化活性均没有明显改变。 siRNA2转染入MDCK细胞48h后，MDCK细胞中的GlyRα1的蛋白表达水平明显下降达60％以上，达到实验的要求。处于ATP耗竭状态1h后，Western blot显示正常组和空转组中，Gly保护组与ATP耗竭组相比，ERK1／2、AKT的磷酸化活性增强，p38MAPK的磷酸化活性减弱，JNK1／2的磷酸化活性没有变化；而在干扰组中，Gly保护组与ATP耗竭组细胞相比ERK1／2、p38MAPK、JNK1／2和AKT的磷酸化活性均无明显变化。结论： ERK1／2、p38MAPK、AKT是甘氨酸受体细胞保护作用的下游途径。甘氨酸与GlyR结合以后，，通过增加ERK1／2、AKT的磷酸化活性；同时抑制p38MAPK的磷酸化活性，从而发挥保护细胞的作用。
[Abstract]:Objective:. To elucidate the downstream signaling pathway of glycine receptor glycine receptor (Glycine receptor GlyR) mediated glycine cell protection, to elucidate the molecular mechanism of glycine antagonism against ATP depletion, and to provide a theoretical basis for the discovery of novel cytoprotective agents. Methods:. In this study, the model of ATP depletion was established by using respiratory chain inhibitor combined with glucose free medium. The canine renal cell line Madin-Darby canine kidneyn MDCK was divided into ATP depletion group and Glycine Glycine protection group. The phosphorylation activity of ERK1 / 2 p38MAPK1 / 2 and AKT were detected by Western blot. The release rate of LDH was observed by ERK1/2 and AKT blocker PD98059 / LY294002. The eukaryotic expression vector of pcDNA3.1-GlyRal was constructed. Liposome-mediated transfection technique was used to transfect the plasmid into human embryonic kidney 293HEK293, and then G418 was used to screen stable cell lines. The stable expression cell lines and wild-type HEK293 cells were subjected to ATP depletion and Gly protection. Western blot was used to detect the phosphorylation activity of ERK1 / 2 p38MAPK1 / 2 and AKT. SiRNA2, which down-regulated the expression of GlyRal in MDCK cells, was transfected into MDCK cells by liposome method, and the cells were exposed to ATP depletion at 48 hours after transfection. Western blot was used to observe the inhibitory effect of siRNA2 on GlyR 伪 1 expression, and Western blot was used to detect the phosphorylation activity of ERK1 / 2 p38MAPK1 / 2 and AKT. Results:. MDCK cells were exposed to ATP depletion for 15 min, 30 min and 1 h for 2 h. The phosphorylation activity of p38 MAPK was decreased in gly protected group compared with ATP depletion group. However, the phosphorylation activity of JNK1/2 did not change. The release rate of LDH in the gly protected group was higher than that in the control group after the addition of PD98059 and LY294002, respectively. HEK293 cells and wild-type HEK293 cells expressing stably GlyR 伪 1 were exposed to ATP depletion for 1 h. The phosphorylation activity of ERK1 / 2AKT in the stable expression cell line was significantly increased compared with that in the ATP depletion group. The phosphorylation activity of p38 MAPK decreased by 1 / 2 of JNK1 / 2 in the stable expression cell line. In wild type HEK293 cells, the phosphorylation activities of gly protection group and ATP depletion group were not significantly changed compared with ERK 1 / 2 p38 MAPK 1 / 2 and AKT. After siRNA2 transfection into MDCK cells for 48 h, the expression level of GlyR 伪 1 in MDCK cells decreased significantly by more than 60%. After 1 hour of ATP depletion, Western blot showed that the phosphorylation activity of ERK1 / 2AK in the gly protected group increased the phosphorylation activity of p38 MAPK and decreased the phosphorylation activity of JNK1 / 2 in the normal group and the idle group compared with the ATP depletion group. However, the phosphorylation activities of ERK1 / 2 p38MAPK1 / 2 and AKT in Gly protected group were not significantly changed compared with those in ATP depletion group. Conclusion:. ERK1 / 2 p38 MAPK- AKT is the downstream pathway of glycine receptor cell protection. After glycine binds with GlyR, it can protect cells by increasing the phosphorylation activity of ERK1 / 2kT and inhibiting the phosphorylation of p38MAPK.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

【引证文献】