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研究生:陳應祈
研究生(外文):Chen, Ying-Chi
論文名稱:YM-1在小鼠發育時期表現之研究
論文名稱(外文):Developmental expression of YM-1 in mice
指導教授:張南驥
指導教授(外文):Chang, Nan-Chi
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:1997
畢業學年度:85
語文別:中文
論文頁數:43
中文關鍵詞:微生物學免疫學YM-1
外文關鍵詞:MICROBIOLOGYIMMUNOLOGY
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以寄生蟲感染小鼠,可刺激腹腔滲出細胞活化,分泌一結晶性蛋白質,命名為YM-1。在正常小鼠骨髓亦可偵測到YM-1的表現。將此蛋白純化可知其分子量約45kDa。利用西方轉漬法偵測懷孕18.5天(E 18.5)小鼠胚胎的多種器官,結果在肝臟及脾臟,發現有顯著YM-1表現;另外在腎臟及肺,亦可偵測到少量。隨後即針對不同時期:懷孕13.5天,16.5天到18.5天,即E13.5,E16.5及E18.5,到出生後(出生當天,出生後七天及十四天及成鼠,即P0,P7,P14,adult)各種組織進行西方轉漬分析。結果顯示YM-1會表現在E16.5的肝臟、脾臟;P0肝臟、脾臟和肺臟,及腎臟有少量;P7的肝臟、脾臟和肺臟;P14的脾臟及肺臟;至成鼠則在骨髓,脾臟及肺部可偵測到YM-1。
發現特定時期的肝臟、肺臟、脾臟可偵測到YM-1蛋白質的表現後,進一步探討這些器官在不同時期表現量的變化。經西方轉漬分析的結果,發現肝臟在E13.5尚無法偵測到YM-1,但E14.5、E16.5時YM-1逐漸增多,到E18.5,P0時達最高峰。P7時漸下降,而P14,成鼠僅能偵測到極低量的YM-1蛋白質。肺臟在E16.5時YM-1的表現量很低,隨後除P14表現量特別低之外,YM-1的量逐漸增多,至成鼠時達最高峰。脾臟則持續有YM-1蛋白質的表現,出生後表現量比出生前略高。
為進一步在轉錄層次確定YM-1的表現,取肝臟、肺臟、脾臟全量RNA進行北方轉漬反應,以YM-1 cDNA片段(P1-P8)作探針,偵測YM-1 mRNA。在肝臟自E16.5起即可雜合到YM-1的mRNA,且大小與PEC全量RNA中所雜合到的相同。在E18.5時,YM-1訊息達最高峰,至P0,P7漸下降,而在P14及成鼠僅能偵測到極低量的YM-1 mRNA。肺臟在E16.5無法偵測到YM-1訊息,隨後除P7量特別低之外,E18.5,P0,P14至成鼠逐漸上升。在成鼠時YM-1訊息達最高峰。在E16.5與E18.5的脾臟中可偵測到少量YM-1訊息,出生後表現量增高。
取E18.5及成鼠的組織進行冷凍切片,用抗YM-1多源抗體及PAP染色法,偵測YM-1在原位(in situ)表現之狀況。在E18.5的肝臟切片,觀察到YM-1陽性細胞均勻分佈,而成鼠卻未偵測到。在肺部的狀況則相反,在E18.5並未偵測到YM-1,但在成鼠肺泡的結構上及細胞間隙中,則可觀察到多量的YM-1表現。在成鼠脾臟的red pulp中,發現有成群的YM-1陽性細胞分佈,但white pulp則無。在胸腺無論是E18.5或成鼠,均未觀察到YM-1的表現。在E18.5的結締組織和腎臟,也觀察到YM-1的表現。在這時期的結締組織中,可觀察到YM-1陽性細胞。在腎臟內,YM-1則出現在皮質部的彎管構造(convoluted tubules)上。
綜合上述結果,正常小鼠除造血器官骨髓,胚胎之肝臟及脾臟,會有YM-1之表現外,另外在成鼠的肺臟,脾臟及胚胎時的腎臟,結締組織,亦有YM-1的表現。除了與造血功能有關連外,YM-1在生長、發育時,可能也扮演了某些角色,這些都必須更進一步的研究。
Activated murine peritoneal macrophages were found to synthesize and secret a protein YM-1 of 43 kDa, which is a non-glycosylated single chain polypeptide. Full length cDNA clones containing the entire open reading frame (ORF) were obtained. It was revealed that YM-1 is composed of 398 amino acids with a leader signal peptide of 21 amino acids. Expression of recombinant YM-1 in baculoviral, mammalian and bacterial systems were successful. Despite the presence of homology with some glycolytic enzymes, no activity was found associated with YM-1. Definitive function of YM-1 awaits further investigation.
By indirect immunofluorescence method, it was observed that YM-1 positive cells appear in bone marrow and lung of normal adult mice. During mouse embryogenesis, hematopoietic events begins in the yolk sac, shifts to fetal liver and later in bone marrow. Western blot analyses of various tissues during developmental stages revealed the expression of YM-1 in fetal liver and spleen on gestation day 18.5 (E18.5). It was also confirmed that YM-1 protein is expressed in adult lung and bone marrow. The total proteins of liver, lung and spleen at various stages of development were analyzed by Western blot.
In liver, expression of YM-1 reaches the peak on E18.5, then decreases to lower level at adult stage. On the other hand, expression of YM-1 in lung increases along the developmental stages. Significant level of YM-1 expression is reached at adult stage. In spleen, YM-1 is detectable in all stages.
Northern blot analyses were employed for examining the transcription of YM-1 in liver, spleen and lung. Although not detectable at E13.5 and E14.5, YM-1 messages appear in liver from E16.5 on. The expression of YM-1 is elevated to a higher at E18.5. Transcription level of YM-1 declines after the day of birth (P0). In lung, transcription of YM-1 is not detectable until E18.5, gradually increases during development and reaches a higher level in adult. Transcription of YM-1 is detectable from development through adulthood.
Tissues from embryo and adult mice were also examined by immunocyto- chemistry. YM-1 expression is observed in cells of fetal liver on E18.5, while no such cells were detected in normal adult liver. Expression of YM-1 was also observed in alveolar structure of adult lung, suggests binding of YM-1 to some extracellular matrix. During embryonic stages, despite its absence in thymus, YM-1 may be detected on convoluted tubules of kidney and in connective tissues. The physiological implication of such distribution is currently not known.

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