本文選題：甘氨酸受體　切入點：ATP耗竭　出處：《南京醫(yī)科大學》2007年碩士論文　論文類型：學位論文

【摘要】： 目的： 明確甘氨酸受體(Glycine receptor，GlyR)介導甘氨酸細胞保護作用的下游信號通路，闡明甘氨酸拮抗細胞ATP耗竭性損傷的分子機制，為發(fā)現(xiàn)新型細胞保護劑提供理論依據(jù)。 方法： 本研究首先使用呼吸鏈抑制劑聯(lián)合無糖培養(yǎng)基共同作用復制細胞ATP耗竭模型。將犬腎細胞(Madin-Darby canine kidney，MDCK)分為ATP耗竭組和Gly(Glycine)保護組，運用Western blot檢測ERK1／2、p38MAPK、JNK1／2和AKT的磷酸化活性變化。分別運用ERK1／2和AKT的阻斷劑PD98059、LY294002，觀察各組LDH釋放率的變化。將構(gòu)建好的pcDNA3.1-GlyRal真核表達載體，應用脂質(zhì)體介導的轉(zhuǎn)染技術(shù)將該質(zhì)粒導入野生型人胚腎細胞(Humanembryonic kidney 293，HEK293)，再用G418篩選穩(wěn)定的細胞表達系。將穩(wěn)定表達細胞系和野生型HEK293細胞處于ATP耗竭和Gly保護狀態(tài)下，運用Western blot檢測ERK1／2、p38MAPK、JNK1／2和AKT的磷酸化活性變化。選用使MDCK細胞中GlyRal表達下調(diào)最明顯的siRNA2，脂質(zhì)體法轉(zhuǎn)染入MDCK細胞，于轉(zhuǎn)染后48小時使其處于ATP耗竭狀態(tài)，運用Western blot觀察siRNA2對GlyRα1表達的抑制效果，并運用Western blot檢測ERK1／2、p38MAPK、JNK1／2和AKT的磷酸化活性變化。 結(jié)果： MDCK細胞處于ATP耗竭狀態(tài)下15min、30min、1h、2h后，Gly保護組與ATP耗竭組相比，ERK1／2、AKT的磷酸化活性均增強，p38MAPK的磷酸化活性減弱，而JNK1／2的磷酸化活性沒有變化。分別加入阻斷劑PD98059和LY294002以后，Gly保護組的LDH釋放率與對照組相比均有升高。 將穩(wěn)定表達GlyRα1的HEK293細胞和野生型HEK293細胞處于ATP耗竭狀態(tài)1h。穩(wěn)定表達細胞系中，Gly保護組與ATP耗竭組相比，ERK1／2、AKT的磷酸化活性均增強，p38MAPK的磷酸化活性減弱，JNK1／2的磷酸化活性沒有改變。野生型HEK293細胞中，Gly保護組和ATP耗竭組相比，ERK1／2、p38MAPK、JNK1／2和AKT的磷酸化活性均沒有明顯改變。 siRNA2轉(zhuǎn)染入MDCK細胞48h后，MDCK細胞中的GlyRα1的蛋白表達水平明顯下降達60％以上，達到實驗的要求。處于ATP耗竭狀態(tài)1h后，Western blot顯示正常組和空轉(zhuǎn)組中，Gly保護組與ATP耗竭組相比，ERK1／2、AKT的磷酸化活性增強，p38MAPK的磷酸化活性減弱，JNK1／2的磷酸化活性沒有變化；而在干擾組中，Gly保護組與ATP耗竭組細胞相比ERK1／2、p38MAPK、JNK1／2和AKT的磷酸化活性均無明顯變化。 結(jié)論： ERK1／2、p38MAPK、AKT是甘氨酸受體細胞保護作用的下游途徑。甘氨酸與GlyR結(jié)合以后，，通過增加ERK1／2、AKT的磷酸化活性；同時抑制p38MAPK的磷酸化活性，從而發(fā)揮保護細胞的作用。
[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 偽 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 偽 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 偽 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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R363

【引證文獻】

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1 張寶s

本文編號：1624946