本文選題：甘氨酸受體 + ATP耗竭　； 參考：《南京醫(yī)科大學》2005年博士論文

【摘要】：目的: 明確細胞ATP耗竭時,甘氨酸對細胞的保護作用是否由甘氨酸受體(glycine receptor, GlyR)所介導;觀察細胞在ATP耗竭-“復能”過程中,其代謝、增殖活性變化的特點。為進一步揭示甘氨酸保護作用機制提供研究基礎和新的思路。 方法: GlyR的配基對ATP耗竭細胞的保護作用。構建甘氨酸受體α1亞單位(GlyRα1)的真核表達載體pcDNA3.1(b),采用脂質(zhì)體法(LipofectAMINETM 2000)將其與pEGFP-C1質(zhì)粒共轉染入缺乏天然GlyR的人胚胎腎細胞株(Human Embryonic Kidney 293,HEK 293)內(nèi),流式細胞儀、激光共聚焦顯微鏡檢測細胞轉染率,分別以RT-PCR及Western blot檢測GlyRα1的mRNA轉錄及蛋白表達。野生型及轉染GlyR的HEK293細胞處于ATP耗竭狀態(tài),觀察甘氨酸、士的寧對細胞的保護作用。犬腎細胞MDCK(Madin-Darby Canine Kidney)作為對照細胞。光學顯微鏡及電鏡觀察細胞形態(tài)及超微結構改變。通過檢測細胞乳酸脫氫酶(Lactate dehydrogenase,LDH)釋放率、細胞膜對不同分子量標記復合物(分子量668Da的碘化丙啶、3kDa及70kDa右旋糖苷)通透性反映細胞膜完整性。通過臺盼藍染色觀察細胞活性。
[Abstract]:Aim: to investigate whether the protection of glycine against ATP depletion is mediated by the glycine receptor (glycine receptor, GlyR, and to observe the role of glycine in the process of ATP-depletion. Its metabolism, proliferative activity change characteristic. In order to further reveal the protective mechanism of glycine to provide a basis for further research and new ideas. Methods: the protective effect of GlyR ligand on ATP depletion cells was studied. The eukaryotic expression vector pcDNA3.1 (b), of glycine receptor 偽 1 subunit (GlyR 偽 1) was constructed and co-transfected with pEGFP-C1 plasmid into human embryonic kidney cell line (Human Embryonic Kidney 293) with liposome assay (LipofectAmNETM 2000). The transfection rate of GlyR 偽 1 was detected by laser confocal microscopy, and the mRNA transcription and protein expression of GlyR 偽 1 were detected by RT-PCR and Western blot, respectively. Wild type and GlyR transfected HEK293 cells were in ATP depletion state. The protective effects of glycine and strychnine on the cells were observed. MDCK (Madin-Darby Canine Kidney) was used as control cell. The changes of cell morphology and ultrastructure were observed by optical microscope and electron microscope. The release rate of lactate dehydrogenase (LDH) was measured. The permeability of cell membrane to different molecular weight marker complexes (668Da, 3kDa and 70kDa dextran) reflected the integrity of cell membrane. Cell activity was observed by trypan blue staining.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2005
【分類號】：R363

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