朱有田臺灣大學:動物科學技術學研究所王健宇Wang, Chien-YuChien-YuWang2010-05-112018-06-292010-05-112018-06-292009U0001-1708200917204400http://ntur.lib.ntu.edu.tw//handle/246246/182078成熟雌性哺乳動物其乳腺發育分為四個時期：處女期、懷孕期、泌乳期及離乳期。其中由腦垂腺所分泌之泌乳素 (prolactin, PRL) 於乳腺上皮細胞之生長、分化及泌乳扮演重要的角色。於泌乳時，泌乳素能透過結合於泌乳素接受體 (prolactin receptor, PRLR) 之酪胺酸激脢Jak-2磷酸化泌乳素接受體，然後泌乳素接受體磷酸化主要之訊號傳遞分子 (signal transducer and activator of transcription 5a, STAT5a)，磷酸化之STAT5a會從細胞質轉移至細胞核，進而調控 b-酪蛋白啟動子之活性。此外，在Caveolin-1 (Cav-1) 的研究中亦發現，Cav-1剔除的小鼠其乳腺管及泡狀構造有提早成熟、提前泌乳與高度磷酸化STAT5a的現象，並且Cav-1認定為具有負調控Jak-2/STAT5a訊息傳遞之功能。另外在小鼠乳腺中，亦了解泌乳素能透過Ras-MAP Kinase訊息傳遞之路徑負調控Cav-1蛋白之表現。比對山羊與人類、山羊與小鼠之Cav-1啟動子序列，其相似度分別為74%、67%。亦比對山羊與人類、山羊與小鼠 b-酪蛋白基因轉錄起始點上游1683個核酸序列，其相似度分別為46%、49%。由於各物種間序列相似度上的差異，因此本研究之目的想探討山羊Cav-1於不朽化山羊乳腺上皮細胞 (caprine mammary epithelial cells, CMEC) 中之功能是否相似於人類及小鼠。先，透過puromycin的篩選，建立穩定表現人類泌乳素接受體 (human prolactin receptor, hPRLR) 之CMEC (CMEC/hPRLR)。為測定山羊 b-酪蛋白啟動子之活性，構築由山羊 b-酪蛋白啟動子 (-1683 - -1) 驅動表現螢火蟲冷光素之質體，並且為矯正轉型效率，亦構築由thymidine kinase (TK) 啟動子驅動表現珊瑚蟲冷光素之質體，並透過將上述二質體共同轉染入CMEC/hPRLR，再經泌乳素刺激，以建立由泌乳素誘導山羊 b-酪蛋白啟動子之系統。經由此系統，山羊 b-酪蛋白啟動子經泌乳素刺激後能提高36.1倍之活性。為了解泌乳素處理濃度對內源性山羊Cav-1表現之影響，以6種不同最終濃度 (濃度從0至15 ug/mL) 之泌乳素處理CMEC/hPRLR，並且在三個時間點 (處理後24、48及72小時) 抽取細胞總蛋白質，經西方吸漬法分析，內源性山羊Cav-1表現量於泌乳素處理後48及72小時，隨處理濃度之增加逐漸減少。接著為探討山羊Cav-1是否會減少泌乳素訊息傳遞誘導之STAT5a磷酸化，將山羊Cav-1質體與山羊STAT5a質體共同轉染入CMEC/hPRLR中，並以泌乳素刺激，經西方吸漬法分析，山羊STAT5a磷酸化程度明顯下降，此外轉染綠螢光蛋白 (EGFP) 之負控制組，並未影響山羊STAT5a的磷酸化。最後為探討山羊Cav-1是否透過降低山羊STAT5a磷酸化，進而降低山羊 b-酪蛋白啟動子之活性，因此於泌乳素誘導山羊 b-酪蛋白啟動子之系統中額外轉染山羊Cav-1，亦觀察到山羊 b-酪蛋白啟動子活性之降低。以上結果可知，CMEC/hPRLR經泌乳素之處理能減少內源性山羊Cav-1之表現，若外源性提高表現山羊Cav-1則會降低山羊STAT5a之磷酸化及降低山羊 b-酪蛋白啟動子之活性。因此，泌乳素處理CMEC/hPRLR時，其內源性山羊Cav-1表現降低及STAT5a磷酸化程度提高，對於促進不朽化山羊乳腺上皮細胞活化山羊 b-酪蛋白啟動子為重要之調控。Development of the adult female mammary gland encounters four distinct stages: virgin, pregnancy, lactation, and involution. During mammary gland development, prolactin (PRL), a pituitary hormone, mediates mammary epithelial cell growth, differentiation and lactation. The act of prolactin in regulating the activity of b-casein promoter via the activation of prolactin tyrosine kinase associated receptor, and its associated protein kinase, Jak-2. Signal transducer and activator of transcription 5a (STAT5a) is the key signaling molecule transfers the prolactin signal from Jak-2 to b-casein promoter. Once STAT5a phosphorylated by Jak-2, STAT5a translocates from the cytoplasm to the nucleus, and activates b-casein promoter during lactation. Besides, based on the study of Caveolin-1 (Cav-1), its null mice accelerated the development of the labuloalveolar compartment, premature milk production, and hyperphosphorylation of STAT5a. In addition, Cav-1 is identified as a negative regulator of Jak-2/STAT5a signaling pathway. Prolactin down-regulates Cav-1 expression via Ras-MAP Kinase pathway also been identified in mouse mammary epithelial cells.he Cav-1 promoter sequence identity between goat versus human and goat versus mouse was 74% and 67%, respectively. The comparison of 1683 nucleotides upstream the transcription start site of b-casein gene between goat versus human and goat versus mouse the sequence similarity is 46% and 49%, respectively. Because diversity among those sequences, the goal in present study is to understand whether the function of goat Cav-1 in caprine mammary epithelial cell (CMEC) is similar to that in mouse and human.irst of all, stable expression of PRL receptor CMEC cells (CMEC/PRLR) lines were established via puromycin selection. In order to measure the b-casein promoter activity, a plasmid possessed goat b-casein promoter (-1683 – -1) drove firefly luciferase (Fluc) reporter protein was constructed. For equivalent the transfection efficiency, another plasmid possessed TK (thymidine kinase) promoter drove the renilla luciferase (Rluc) reporter protein also been constructed. A system that prolactin mediated b-casein promoter in CMEC/PRLR cells was established by co-expression of b-casein promoter Fluc plasmid and TK promoter Rluc plasmid in the CMEC/PRLR cells in response to the stimulation of PRL. The activity of goat b-casein promoter activity was enhanced 36.1 fold after PRL treatment. To understand the dosage effect of PRL to the endogenous Cav-1 expression, 6 final PRL concentrations (range from 0 to 15 ug/ml) were used to treat the CMEC/PRLR cells, then the cell proteins were harvested at 3 time courses (24, 48, 72 hr post-treatment). The results show that endogenous Cav-1 down-regulated in response to PRL treatment at 48, and 72 hr and dose dependent. To study whether goat Cav-1 could down-regulate the activity (phosphorylation) of STAT5a via PRL signal cascade, the phsphorylayion state of STAT5a were determined with or without exogenous goat Cav-1 protein expression in CMEC/PRLR cells after PRL stimulation. The phosphorylation of STAT5a was decreased after PRL stimulation, furthermore expression of the control protein EGFP (Enhanced Green Fluorescent Proteins) did not alter STAT5a phosphorylation. Finally, to study whether goat Cav-1 could down-regulate the b-casein promoter activity by down-regulate the phosphorylation of STAT5a. We transient transfect gCav-1 in the PRL induce b-casein promoter system. The gCav-1 also down-regulate the b-casein promoter activity.n conclusion, these results showed that PRL down-regulated the endogenous Cav-1 expression, and elevated expression of exogenous Cav-1 decreased the phosphorylation of STAT5a proteins and b-casein promoter activity. Therefore, PRL down-regulated endogenous Cav-1 expression and up-regulated STAT5a phosphorylation is important for improving b-casein promoter activity in CMEC.目錄....................................................II次....................................................VI、前言.................................................1、中文摘要.............................................2、英文摘要.............................................4、文獻檢討.............................................6、活體乳腺構造與發育...................................6. 乳腺構造..............................................6. 乳腺發育..............................................6、調控乳腺發育之內泌素與乳蛋白表現.....................7、影響 b-酪蛋白表現之調控機制..........................9. 胞外基質 (extracellular matrix, ECM) 對 b-酪蛋白基因表現之調控...................................................9. 生太與類固醇激乳內泌素對 b-酪蛋白基因轉錄之調控......10. b-酪蛋白啟動子調控 b-酪蛋白之表現....................11、泌乳素..............................................12. 概況.................................................12. 生理功能.............................................12. 泌乳素於乳腺上皮細胞啟動之訊息傳遞...................13. 負調控泌乳素誘導之JAK/STAT訊息傳遞...................13、Caveolin............................................14. 發現與功能...........................................14. Caveolin-1於乳腺細胞中對 b-酪蛋白表現之影響..........15、材料與方法..........................................17一部份：建立穩定表現人類泌乳素接受體之山羊乳腺上皮細胞株......................................................17-1、細胞培養...........................................17-2、細胞繼代...........................................17-3、細胞轉型感染.......................................17-4、抗生素篩選建立穩定表現外源性蛋白之CMEC細胞株.......18二部份：泌乳素誘導山羊 b-酪蛋白基因啟動子之活性.......19-1、誘導CMEC細胞株活化 b-casein啟動子..................19-2、雙重冷光脢分析系統.................................19三部份：細胞內蛋白質表現之偵測........................19-1、細胞蛋白質萃取.....................................19-2、蛋白質定量.........................................20-3、西方吸漬 (Western blotting)........................20-4、細胞免疫螢光染色...................................21四部份：山羊caveolin-1 cDNA之選殖.....................22-1、細胞total RNA萃取..................................22-2、反轉錄脢聚合鏈鎖反應...............................22-3、聚合脢鏈鎖反應.....................................23-4、質體pGEM-T easy gCav-1與pcDNA4 A gCav-1 myc/His建構......................................................24. 質體pGEM-T easy gCav-1建構...........................24. 質體pcDNA4 A gCav-1 myc/His建構......................25-5、小量質體DNA萃取....................................25-6、中量質體DNA萃取....................................26五部份：腺病毒載體系統之架構..........................27-1、細胞培養...........................................27-2、重組腺病毒載體pAd/CMV/V5-DEST gCav-1與pAd/CMV/V5-DEST EGFP建構................................................27-3、初代重組腺病毒製備.................................29-4、重組腺病毒力價擴增.................................29-5、重組腺病毒力價測試 (Plaque assay)..................29-6、重組腺病毒感染CMEC細胞株MOI (Multipicity of infection) 測試.........................................30六部份：統計分析......................................30、結果................................................32、建立穩定表現人類泌乳素接受體之山羊乳腺上皮細胞株....32、泌乳素處理增加CMEC/hPRLR中外源性山羊 b-酪蛋白基因啟動子活性....................................................34、泌乳素負調控CMEC/hPRLR內源性Caveolin-1表現..........37、山羊caveolin-1 cDNA之選殖...........................37、山羊Caveolin-1蛋白負調控STAT5a磷酸化與 b-酪蛋白啟動子活性......................................................39、表現gCav-1與EGFP腺病毒載體系統之構築................40、討論................................................43、建立穩定表現人類泌乳素接受體之CMEC..................43、泌乳素增加CMEC/hPRLR中外源性山羊 b-酪蛋白基因啟動子活性......................................................44、泌乳素負調控CMEC/hPRLR內源性Caveolin-1蛋白表現......45、表現goat Caveolin-1蛋白抑制STAT5a磷酸化.............46、表現gCav-1與EGFP腺病毒載體系統之構築................47、結論................................................70、參考文獻............................................71錄....................................................78application/pdf1401529 bytesapplication/pdfen-USCaveolin-1乳腺上皮細胞泌乳素酪蛋白啟動子泌乳mammary epithelial cellprolactincasein promoterlactationthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182078/1/ntu-98-R96626014-1.pdf