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研究生:林伯軒
研究生(外文):Bo-Shen Lin
論文名稱:第二型肺泡細胞磷酸甘油酯醯基轉移酶之鑑定分析與特性研究
論文名稱(外文):Identification and characterization of a lysophosphatidylcholine acyltransferase in alveolar type II cells
指導教授:盧志峰盧志峰引用關係
指導教授(外文):Jyh-Feng Lu
學位類別:碩士
校院名稱:輔仁大學
系所名稱:基礎醫學研究所碩士班
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:64
中文關鍵詞:磷酸甘油酯醯基轉移酶第二型肺泡細胞肺泡表面活性劑磷脂質
外文關鍵詞:lysophosphatidylcholine acyltransferasealveolar type II cellssurfactantphospholipid
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摘要
在肺臟中,肺泡為氣體交換的核心部位。肺泡表層主要由第一型肺泡細胞和第二型肺泡細胞建構而成。第二型肺泡細胞主要功能為分泌肺泡表面活性劑以降低肺泡氣液界面的表面張力,避免肺泡塌陷。肺泡表面活性劑由磷脂質與蛋白質組成。磷脂質是由磷脂醯膽鹼,主要為二棕櫚磷脂醯膽鹼,與磷脂甘油酯構成。二棕櫚磷脂醯膽鹼是表面活性劑中最主要用以降低表面張力的成分。二棕櫚磷脂醯膽鹼可以藉由新生合成或是由不飽和磷脂醯膽鹼重塑而產生。重塑路徑是以磷脂酶A2,將不飽和磷脂醯膽鹼分子中的甘油骨架第2號碳位置上的酯化不飽和醯基移除形成1-脂肪酸磷酸甘油酯醯膽鹼。藉由磷酸甘油酯醯基轉移酶(LPCAT)的催化,將一棕櫚磷酸甘油酯醯膽鹼與棕櫚酸輔酶A鍵結重塑為二棕櫚磷脂醯膽鹼。本實驗室之前已發現特定表現於第二型肺泡細胞之基因群,其中之FR1基因可轉譯為具有534個胺基酸,分子量約為59 kDa的蛋白質。胺基酸序列比較分析發現其具有相似於大腸桿菌的1-脂肪酸甘油磷酸酯醯基轉移酶(plsC)的特殊結構(acyltransferase domain),推測FR1所表現的蛋白質可能為第二型肺泡細胞中磷酸甘油酯醯基轉移酶。藉由原核細胞大量表現系統,表現並純化具有麥芽糖結合蛋白與組胺酸標記的磷酸甘油酯醯基轉移酶融合蛋白,再以其作為抗原產生特異性抗體血清後,以親和性管柱層析法加以純化。藉由純化之抗體血清以組織切片進行免疫化學分析、免疫螢光和西方墨點法偵測此蛋白質於肺臟中第二型肺泡細胞的表現情形。再以梯度密度離心方式分離肺臟組織中的胞器,確認此蛋白質位於細胞內質網中,並以雷射掃瞄共軛焦顯微鏡技術驗證此一分佈情形。最後,藉由真核細胞293T細胞表現此蛋白質並分離細胞中的胞器進行磷酸甘油酯醯基轉移酶的活性測試。
The alveolus is the major gas exchange unit in lungs. The alveolar epithelium is mainly composed of two types of cells, alveolar type I and type II cells. The major function of type II cells is to produce pulmonary surfactant. Pulmonary surfactant is secreted from alveolar type II cells to the air–liquid interface where it reduces surface tension and prevents atelectasis of alveoli. Pulmonary surfactant is composed of phospholipids and surfactant proteins. Phospholipids include phosphatidylcholines (PC), mainly dipalmitoylphosphatidylcholine (DPPC), and phosphatidylglycerols. DPPC is most responsible for the surface tension-lowering properties of pulmonary surfactant. Production of DPPC can be achieved by both de novo synthesis and the remodeling pathways. In the remodeling pathway, unsaturated acyl group of cellular phosphatidylcholines is removed by phospholipase A2 resulting in 1-palmitoyl-2-
lysophosphatidylcholines, followed by reacylation of 1-palmitoyl-2-lysophos-
phatidylcholines with palmitoyl-CoA via the catalysis of a lysophosphatidylcholine (lysoPC) acyltransferase (LPCAT). We identified a putative LPCAT whose expression is enriched in alveolar type II cells of Sprague Dawley (SD) rat. The cloned cDNA encodes a protein of 534 amino acids with an estimated molecular mass of 59 kDa. Amino acid sequences of this putative acyltransferase revealed a highly conserved domain similar to 1-acyl-sn-glycerol-3-phosphate acyltransferase (plsC) in Escherichia coli(E. coli). Using prokaryotic expression system, a maltose binding protein and 6x histidine tagged LPCAT was successfully expressed and purified using affinity chromatography. Purified proteins were injected into rabbits for specific anti-sera production. Anti-LPCAT antibodies were also purified from immunized sera by affinity chromatography. Using purified anti-LPCAT antibodies, we localized LPCAT protein to alveolar type II cells by immunohistochemistry, immunoflurosence and Western blotting. Using zonal centrifugation, LPCAT was localized to the microsomal organelle fraction. The result was also confirmed by confocal microscopy. Using eukaryotic 293T cell expression system in combination with zonal centrifugation, the microsomal organelle fractions from transfected cells
were tested for acyltransferase activity.
目錄
摘要………………………………………………………………………………i
英文摘要…………………………………………………………………………iii
致謝………………………………………………………………………………v
目錄………………………………………………………………………………vii
圖表目錄…………………………………………………………………………viii
第一章 緒言………………………………………………………………………1
第二章 文獻回顧…………………………………………………………………2
第三章 材料與方法………………………………………………………………10
第四章 結果………………………………………………………………………25
第五章 討論………………………………………………………………………32
第六章 結論………………………………………………………………………38
第七章 參考資料…………………………………………………………………55
附表一 ……………………………………………………………………………61
附表二 ……………………………………………………………………………62
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