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研究生:王志偉
研究生(外文):Jyhi-Wei Wang
論文名稱:小鼠全腦組織與神經母細胞瘤細胞株之Proliferin表現分析
論文名稱(外文):The Expression Profile of Proliferin in Mouse Brain and Neuroblastoma Cell Line
指導教授:朱有田朱有田引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:畜產學研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:52
中文關鍵詞:全腦組織神經母細胞瘤細胞株小鼠
外文關鍵詞:whole brainNeuro-2aproliferin
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泌乳素基因家族諸多成員之中,proliferin (PLF)為其一員,主要功能為幫助小鼠懷孕時期胎盤與子宮的正常發育。PLF屬醣基化蛋白質,其表現受生長因子之調控。PLF擁有不止一種接受體,cation-independent mannose 6-phosphate receptor (CI-MPR)為已知之接受體,其餘PLF接受體基因尚未被發現。PLF本身為基因家族,有4個家族成員。PLFs相關研究多將焦點放在胎盤與表皮組織,然而,尚有許多其他組織表現PLFs。至目前為止,尚無文獻討論到PLF與腦部的關係。本研究的目的在於探討小鼠腦部之PLF表現與其可能之功能。我們首先發現PLF mRNA表現在成年ICR小鼠之全腦組織中。接著分析發育過程小鼠全腦組織之PLF mRNA表現量,試驗結果顯示小鼠胎兒時期與成年時期腦組織有較高之PLF mRNA表現量,而新生兒時期的表現量相對地較低。分析小鼠繁殖過程全腦組織PLF mRNA表現量的變化,發現分娩當天有較低的表現量。利用診斷式PCR分析小鼠全腦組織所表現的PLF基因家族,發現PLF1和PLF3 mRNA為主要的PLF基因家族成員,無PLF2和PLF4 mRNA表現。小鼠全腦組織的基因選殖試驗中,我們獲得了PLF1、PLF3以及PLF1(-exon3)的cDNA序列,DNA定序結果顯示全腦組織確實有PLF mRNA表現。Neuro-2a是一個來自於成年小鼠腦部的神經母細胞瘤細胞株,以RT-PCR分析其mRNA發現有PLF與CI-MPR基因表現,而PLF基因家族以PLF1、PLF3和PLF4為主。在短暫轉形感染與細胞免疫染色試驗中,發現外源性PLF1蛋白質能表現在Neuro-2a細胞之內質網。觀察細胞外形,表現外源性PLF1之Neuro-2a其細胞表面具有明顯之微絨毛構造。依據本研究結果,我們推測腦部PLF表現可能受到bFGF與糖皮素的調控,於不同腦部發育時期參與神經母細胞之分化。
Proliferin (PLF) (also termed as mitogen-regulated protein, MRP) is a prolactin-like placental hormone that plays an important role in the formation of uteroplacenta. Expression of PLF is temporally regulated in mouse placenta and is found to be relatively less in early-gestation, peaks in mid-gestation and then declines in late-gestation. There are four PLF homologues, PLF1, PLF2, PLF3 and PLF4 in placenta, and different form of PLF also selectively expresses in small intestine, ear and tail. PLF in vitro is found as a secreted glycoproteins from cultured mouse cells in which mitogens like of epidermal growth factor (EGF) and fibroblast growth factor (FGF) regulate their mRNA and protein expression. Cell lines derived from various origins including skin, bone marrow, ascites, fetus fibroblast, and muscular tumor express PLF which have a role in cell proliferation, differentiation and migration. Until now, there is no paper discussing the PLF expression in brain.
Cation-independent mannose 6-phosphate receptor (CI-MPR), designated as insulin-like growth factor II receptor, is a 300 kD membrane bound protein that adopts diverse ligands including proliferin. Intracellularly, CI-MPR binds with its ligands, traffics among trans-golgi network, endosome-lysosomal system and cell surface. CI-MPR serves as a tumor suppressor and is associated with cell growth and motility. PLF-induced endothelial cell migration is mediated by CI-MPR. The expression of CI-MPR is necessary for normal development of various tissues including brain suggesting that PLF may be involved in brain development and function.
  Neuro-2a is a neuroblastoma cell line isolated from adult mouse brain tumor. Using RT-PCR, we examined the PLF mRNA expressing in Neuro-2a. We addressed whether PLF expresses in brain. Results indicated that PLF mRNA expresses in both male and female adult ICR mouse brains but comparatively less in postnatal day 13. According to semi-quantitative RT-PCR, PLF mRNA levels relative to G3PDH internal control in embryonic day 14, postnatal day 0, 7, 14, 70, gestation day 7, 14, lactation day, 0, 7, 14 and weaning day 3 were 1.139, 0.03, 0, 0.839, 2.215, 0.842, 0.791, 0.215, 1.133, 0.751 and 0.718 respectively. It was found that the level was relatively high in embryonic and adult days compared to postnatal days. PLF1 and PLF3 are the major species found in brain, while PLF1, PLF3 and PLF4 were prominent in Neuro-2a. In addition to PLF1 and PLF3, PLF1 minus exon3 cDNA were also cloned from mouse brain, suggesting that there are alternative splicing forms in mouse brain. To study the effect of PLF in Neuro-2a, we constructed PLF1 into the eukaryotic expressing vector. After confirming the expression of CI-MPR mRNA, we introduced the vector into Neuro-2a. Results showed that exogenous PLF1 colocalized on the endoplasmic reticulum and may be associated with the formation of microvilli on the cell surface of Neuro-2a. Because microvilli forming on the cell sufrace of resting and differentiating cell, PLF1 ehanced the microvilli formation of Neuro-2a suggests that exogenous PLF1 promote the differentiation of the neuroblastoma cell line Neuro-2a.
  Interestingly, EGF and FGF are strongly involved in brain development as well as proliferation and migration of neuronal progenitor. On the basis of our data in brain, we propose that PLF takes part differentially in neuroblast differentiation during various stages of brain development.
壹、摘要………………………………………………………………………………1
貳、前言………………………………………………………………………………2
參、文獻檢討
一、Proliferin基因的發現與基因序列之分析
(一) 發現與命名……………………………………………………………....4
(二) cDNA序列、蛋白質結構與蛋白質表現……………………….………5
(三) PLF與生乳素之關聯性……………………………………….…………6
(四) PLF家族…………………………………………………………………7
(五) 啟動子序列分析…………………………………………….…………...8
二、Proliferin在懷孕中所扮演的角色
(一) 胎盤構造………………………………………………………..……….9
(二) 胎盤PLF表現…………………………………………………………..9
(三) 發育調控…………………………………………………………….…10
(四) 接受體之組織分布…………………………………………………….10
(五) 血管生成…………………………………………………………….…11
(六) 子宮增生……………………………………………………………….11
三、Proliferin作為細胞傳訊物質
(一) PLF與細胞增生………………………………………………………...12
(二) PLF與細胞移動…………………………………………………….......13
肆、材料與方法
一、小鼠飼養……………………………………………………………..……..14
二、小鼠之犧牲與腦組織採樣…………………………………………………14
三、萃取腦組織之total RNA……………..……………………………………14
四、反轉錄………………………………………………....……………………15
五、聚合酶鏈鎖反應……………………………………………………………15
六、診斷式聚合酶鏈鎖反應……………………………………………………17
七、DNA接合反應……………………………...………………………………17
八、細菌電穿孔轉形法…………………………………………..……………..18
九、快速篩選菌落…………………………………...………………………….19
十、萃取及純化小量質體DNA………………………..………………………19
十一、存菌與活化………………………………………………...…………….20
十二、自洋菜膠內回收DNA……………………………….………………….20
十三、萃取及純化中量質體DNA………………………….………………….21
十四、細胞解凍……………………………...………………………………….22
十五、細胞培養…………………………………..……………………………..22
十六、細胞繼代………………………………...……………………………….22
十七、細胞冷凍儲存………………………………..…………………………..23
十八、細胞轉形感染……………………………………………………………23
十九、細胞免疫染色……………………………………………………………23
伍、結果與討論
一、成年公、母小鼠腦組織中表現PLF mRNA……………………………25
二、不同發育期小鼠腦組織表現之PLF mRNA……………...……………25
三、不同繁殖期小鼠腦組織表現之PLF mRNA……………………………27
四、PLF1與PLF3為小鼠腦組織所表現之PLF基因家族成員……….…29
五、自小鼠腦組織選殖PLF1、PLF3與PLF1(-eoxn3) cDNA….…………31
六、PLF1(-exon3)之胺基酸序列分析………………………….……………33
七、Neuro-2a細胞表現內源性PLF mRNA……………………...…………33
八、Neuro-2a細胞所表現的PLF基因家族成員………………....…………36
九、小鼠腦組織與Neuro-2a細胞皆有CI-MPR mRNA表現…...…………36
十、外源性PLF1蛋白質能夠表現在Neuro-2a細胞之內質網………...…40
陸、參考文獻……………………………………………………………………...…45
柒、英文摘要………………………………………………………………………...51
Albert, B., D. Bray, J. Lewis, M. Raff, K. Roberts and J. D. Watson. 1989. Molecular biology of the cell, 2nd edition. Garland Publishing, Inc., New York & London, pp.311, 794~802.
Caldwell, M. A., X. He, N. Wilkie, S. Pollack, G. Marshall, K. A. Wafford, and C. N. Svendsen. 2001. Growth factors regulate the survival and fate of cells derived from human neurospheres. Nat. Biotechnol. 19: 475-479.
Choong, M. L., A. C. L. Tan, B. Luo and H. F. Lodish. 2003. A novel role for proliferin-2 in the ex vivo expansion of hematopoietic stem cells. FEBS Lett. 550: 155-162.
Connor, A. M., P. Waterhouse, R. Khokha, and D. T. Denhardt. 1989. Characterization of a mouse mitogen-regulated protein/proliferin gene and its promoter: A member of the growth hormone/prolactin gene superfamily. Biochim. Biophys. Acta. 1009: 75-82.
Corbacho, A. M., G. Martinez de la Escalera, and C. Clapp. 2002. Roles of prolactin and related members of the prolactin/growth hormone/placental lactogen family in angiogenesis. J. Endocrinol. 173: 219-238.
Dahms, N. M. and M. K. Hancock. 2002. P-type lectins. Biochim. Biophys. Acta 1572: 317-340.
Deininger, P. and W. Smith. 1987. A rapid screening procedure for the identification of recombinant bacterial clones. Biotech. 5: 11-13.
Diamond, M. I., J. N. Miner, S. K. Yoshinaga, and K. R. Yamamoto. 1990. Transcription factor interactions: Selectors of positive or negative regulation from a single DNA element. Science 249: 1266-1272.
Dono, R. 2003. Fibroblast growth factors as regulators of central nervous system development and function. Am. J. Physiol. Regul. Integr. Comp. Physiol. 284: R867-R881.
Dono, R., G. Texido, R. Dussel, H. Ehmke, and R. Zeller. 1998. Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice. EMBO J. 17: 4213-25.
Duckworth, M. L., L. M. Peden, and H. G. Friesen. 1986. Isolation of a novel prolactin-like cDNA clone from developing rat placenta. J. Biol. Chem. 261: 10879-10884.
Fang, Y., P. Lepont, J. T. Fassett, S. P. Ford, A. Mubaidin, R. T. Hamilton, and M. Nilsen-Hamilton. 1999. Signaling between the placenta and the uterus involving the mitogen-regulated protein/proliferins. Endocrinol. 140: 5239-5249.
Fassett, J. T. and M. Nilsen-Hamilton. 2001. Mrp3, a mitogen-regulated protein/proliferin gene expressed in wound healing and in hair follicles. Endocrinol. 142: 2129-2137.
Fassett, J. T., R. T. Hamilton, and M. Nilsen-Hamilton. 2000. Mrp4, a new mitogen-regulated protein/proliferin gene; unique in this gene family for its expression in the adult mouse tail and ear. Endocrinol. 141: 1863-1871.
Ghosh, P., N. M. Dahms, and S. Kornfeld. 2003. Mannose 6-phosphate receptors: New twists in the tale. Nature Rev. 4: 202-212.
Gil-Torregrosa, B., J. L. Urdiales, J. Lozano, J. M. Mates, and F. Sanchez-Jimenez. 1994. Expression of different mitogen-regulated protein/proliferin mRNAs in ehrlich carcinoma cells. FEBS lett. 349: 343-348.
Groskopf, J. C. and D. I. H. Linzer. 1994. Characterization of a delayed early serum response region. Mol. Cell. Biol. 14: 6013-6020.
Groskopf, J. C., L.-J. Syu, A. R. Saltiel, and D. I. H. Linzer. 1997. Proliferin induces endothelial cell chemotaxis through a G protein-coupled, mitogen-activated protein kinase-dependent pathway. Endocrinol. 138: 2835-2840.
Hafez, B. and E. S. E. Hafez. 2000. Reproduction in farm animals, 7th edition. Lippincott Williams& Wilkins, A Wolters Kluwer Company, USA, pp.132.
Hawkes, C. and S. Kar. 2003. Insulin-like growth factor-II/mannose-6-phosphate receptor: Widespread distribution in neuros of the central nervous system including those expressing cholinergic phenotype. J. Compara. Neurol. 458: 113-127.
Jackson, D. and D. I. H. Linzer. 1997. Proliferin transport and binding in the mouse fetus. Endocrinol. 138: 149-155.
Jackson, D., O.V. Volpert, N. Bouck, and D. I. H. Linzer. 1994. Stimulation and inhibition of angiogenesis by placental proliferin and proliferin-related protein. Science. 266: 1581-1584.
Jackson-Grusby, L. L., D. Pravtcheva, F. H. Ruddle, D. I. H. Linzer. 1988. Chromosomal mapping of the prolactin/growth hormone gene family in the mouse. Endocrinol. 122: 2462-6.
Kang, Y., J. S. Park, S. H. Kim, Y. J. Shin, W. Kim, H.-J. Joo, J.-S. Chun, H. H. Kim, and M. J. Ha. 2000. Overexpression of protein kinase Cd represses expression of proliferin in NIH3T3 cells that regulates cell proliferation. Mol. Cell Biol. Res. Commun. 4: 181-187.
Kerppola, T. K., D. Luk and T. Curran. 1993. Fos is a preferential target of glucocorticoid receptor inhibition of AP-1 activity in vitro. Mol. Cell. Biol. 13: 3782-3791.
Lange, K. 1999. Microvillar Ca++ signaling: A new view of an old problem. J. Cell. Physiol. 180: 19-34.
Lau, M. M., C. E. Stewart, Z. Liu, H. Bhatt, P. Rotwein and C. L. Stewart. 1994. Loss of the imprinted IGF2/cation-independent mannose 6-phosphate receptor results in fetal overgrowth and perinatal lethality. Genes Dev. 8: 2953-2963.
Lee, S.-J. and D. Nathans. 1987. Secretion of proliferin. Endocrinol. 120: 208-213.
Lee, S.-J. and D. Nathans. 1988. Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors. J. Biol. Chem. 263: 3521-3527.
Lee, S.-J., F. Talamantes, E. Wilder, D. I. H. Linzer, and D. Nathans. 1988. Trophoblastic giant cells of the mouse placenta as the site of proliferin synthesis. Endocrinol. 122: 1761-1768.
Linzer, D. I. H. and D. Nathans. 1984. Nucleotide sequence of a growth-related mRNA encoding a member of the prolactin-growth hormone family. Proc. Natl. Acad. Sci. USA. 81: 4255-4259.
Linzer, D. I. H. and J. C. Mordacq. 1987. Transcriptional regulation of proliferin gene expression in response to serum in transfected mouse cells. EMBO J. 6: 2281-2288.
Linzer, D. I. H. and S. J. Fisher. 1999. The placenta and the prolactin family of hormones: Regulation of the physiology of pregnancy. Mol. Endocrinol. 13: 837-840.
Linzer, D. I. H., S.-J. Lee, L. Ogren, F. Talamantes, and D. Nathans. 1985. Identification of proliferin mRNA and protein in mouse placenta. Proc. Natl. Acad. Sci. USA. 82: 4356-4359.
Ma, G. T., M. E. Roth, J. C. Groskopf, F.-Y. Tsai, S. H. Orkin, F. Grosveld, J. D. Engel, and D. I. H. Linzer. 1997. GATA-2 and GATA-3 regulate trophoblast-specific gene expression in vivo. Development 124: 907-914.
Malkoski, S. P. and R. I. Dorin. 1999. Composite glucocorticoid regulation at a functionally defined negative glucocorticoid response element of the human corticotropin-releasing hormone gene. Mol. Endocrinol. 13: 1629-1644.
Malyankar, U. M., S. R. Rittling, A. Connor, and D. T. Denhardt. 1994. The mitogen-regulated protein/proliferin transcript is degraded in primary mouse embryo fibroblast but not 3T3 nuclei: Altered RNA processing correlates with immortalization. Proc. Natl. Acad. Sci. USA. 91: 335-339.
Mohideen, M.-A. P. K., A. Hruska-Hageman, and M. Nilsen-Hamilton. 1999. A unique bFGF-responsive transcriptional element. Gene 237. 81-90.
Mordacq, J. C. and D. I. H. Linzer. 1989. Co-localization of elements required for phorbol ester stimulation and glucocorticoid repression of proliferin gene expression. Genes Dev. 3: 760-769.
Muscat, G. E., K. Gobius and J. Emery. 1991. Proliferin, a prolactin/growth formone-like peptide represses myogenic-specific transcription by the suppression of an essential serum response factor-like DNA-binding activity. Mol. Endocrinol. 5: 802-814.
Nelson, J. T., N. Rosenzweig, and M. Nilsen-Hamilton. 1995. Characterization of the mitogen-regulated protein (proliferin) receptor. Endocrinol. 136: 283-288.
Nilsen-Hamilton, M., J. M. Shapiro, S. L. Massoglia, and R. T. Hamilton. 1980. Selective stimulation by mitogens of incorporation of 35S-methionine into a family of proteins released into the medium by 3T3 cells. Cell 20: 19-28.
Nilsen-Hamilton, M., Y. J. Jang, E. Alvarez-Azaustre, and R. T. Hamilton. 1988. Regulation of the production of a prolactin-like protein (MRP/PLF) in 3T3 cells and in the mouse placenta. Mol. Cell Endocrinol. 56: 179-190.
Parfett, C. L. J., R. T. Hamilton, B. W. Howell, D. R. Edwards, M. Nilsen-Hamilton, and D. T. Denhardt. 1985. Characterization of a cDNA clone encoding murine mitogen-regulated protein: Regulation of mRNA levels in mortal and immortal cell lines. Mol. Cell. Biol. 5: 3289-3292.
Schaffer, B. S., M.-F. Lin, J. C. Byrd, J. H. Y. Park and R. G. MacDonald. 2003. Opposing roles for the insulin-like growth factor (IGF)-II and mannose 6-phosphate (Man-6-P) binding activities of the IGF-II/Man-6-P receptor in the growth of prostate cancer cells. Endocrinol. 144: 955-966.
Soares, M. J., H. Muller, K. E. Orwig, T. J. Peters, and G. Dai. 1998. The uteroplacental prolactin family and pregnancy. Bio. Reprod. 58: 273-284.
Toft, D. J., S. B. Rosenberg, G. Bergers, O. Volpert, and D. I. H. Linzer. 2001. Reactivation of proliferin gene expression is associated with increased angiogenesis in a cell culture model of fibrosarcoma tumor progression. Proc. Natl. Acad. Sci. USA. 98: 13055-13059.
Volpert, O., D. Jackson, N. Bouck, and D. I. H. Linzer. 1996. The insulin-like growth factor II/mannose 6-phosphate receptor is required for proliferin-induced angiogenesis. Endocrinol. 137: 3871-3876.
Wiemers, D. O., L. Shao, R. Ain, G. Dai, and M. J. Soares. 2003. The mouse prolactin gene family locus. Endocrinol. 144: 313-325.
Wilder, E. L. and D. I. H. Linzer. 1986. Expression of multiple proliferin genes in mouse cells. Mol. Cell. Biol. 6: 3283-3286.
Wilder, E. L. and D. I. H. Linzer. 1989. Participation of multiple factors, including proliferin, in the inhibition of myogenic differentiation. Mol. Cell. Biol. 9: 430-441.
Yamaguchi, M, T. Imai, T. Maeda, M. Sakata, A. Miyake and D. I. H. Linzer. 1995. Cyclic adenosine 3’,5’-monophosphate stimulation of placental proliferin and proliferin-related protein secretion. Endocrinol. 136:2040-2046.
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