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  • 學位論文

小鼠全腦組織與神經母細胞瘤細胞株之Proliferin表現分析

The Expression Profile of Proliferin in Mouse Brain and Neuroblastoma Cell Line

指導教授 : 朱有田
共同指導教授 : 姜延年

摘要


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。

並列摘要


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.

並列關鍵字

whole brain Neuro-2a proliferin

參考文獻


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