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

【摘要】：目的: 明確細(xì)胞ATP耗竭時(shí),甘氨酸對(duì)細(xì)胞的保護(hù)作用是否由甘氨酸受體(glycine receptor, GlyR)所介導(dǎo);觀察細(xì)胞在ATP耗竭-“復(fù)能”過程中,其代謝、增殖活性變化的特點(diǎn)。為進(jìn)一步揭示甘氨酸保護(hù)作用機(jī)制提供研究基礎(chǔ)和新的思路。 方法: GlyR的配基對(duì)ATP耗竭細(xì)胞的保護(hù)作用。構(gòu)建甘氨酸受體α1亞單位(GlyRα1)的真核表達(dá)載體pcDNA3.1(b),采用脂質(zhì)體法(LipofectAMINETM 2000)將其與pEGFP-C1質(zhì)粒共轉(zhuǎn)染入缺乏天然GlyR的人胚胎腎細(xì)胞株(Human Embryonic Kidney 293,HEK 293)內(nèi),流式細(xì)胞儀、激光共聚焦顯微鏡檢測(cè)細(xì)胞轉(zhuǎn)染率,分別以RT-PCR及Western blot檢測(cè)GlyRα1的mRNA轉(zhuǎn)錄及蛋白表達(dá)。野生型及轉(zhuǎn)染GlyR的HEK293細(xì)胞處于ATP耗竭狀態(tài),觀察甘氨酸、士的寧對(duì)細(xì)胞的保護(hù)作用。犬腎細(xì)胞MDCK(Madin-Darby Canine Kidney)作為對(duì)照細(xì)胞。光學(xué)顯微鏡及電鏡觀察細(xì)胞形態(tài)及超微結(jié)構(gòu)改變。通過檢測(cè)細(xì)胞乳酸脫氫酶(Lactate dehydrogenase,LDH)釋放率、細(xì)胞膜對(duì)不同分子量標(biāo)記復(fù)合物(分子量668Da的碘化丙啶、3kDa及70kDa右旋糖苷)通透性反映細(xì)胞膜完整性。通過臺(tái)盼藍(lán)染色觀察細(xì)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(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R363

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