https://scholars.lib.ntu.edu.tw/handle/123456789/159897
標題: | Proliferin 1生物特性之探討 Biological Characterization of Proliferin 1 |
作者: | 謝桓裕 Hsieh, Heng-Yu |
關鍵字: | 分泌性醣化蛋白質;PLF1 | 公開日期: | 2004 | 摘要: | Proliferin(PLF)在泌乳素及生長激素的家族成員中,屬於分泌性醣化蛋白質。已知在纖維母細胞有PLF1表現,但會抑制肌肉細胞分化。PLF1能夠促進細胞增殖,並選擇性地抑制多種調控肌肉細胞分化的基因之轉譯,先前我們發現在脂肪細胞的分化過程中PLF1 mRNA 的表現有差異性,並闡述說明PLF蛋白質在生物系統中的角色,包括:脂肪細胞生成作用、體重之調整、癌細胞的增殖及轉移,我們研究的目的主要為重組蛋白表現、多株抗體的製造及不同生物系統中PLF1蛋白質的表現量的評估。 我們選殖PLF1 cDNA片段構築於細菌的表現質體,發現蛋白質的表現為一明顯之不溶解性蛋白質,存在包涵體內。我們溶解此一蛋白質,且完成蛋白質再摺疊,成母繸o有效的蛋白質,利用溶解性蛋白質經由脾臟注射方式使兔子產生免疫反應來獲得抗體。接著進行抗體效價檢測,利用重組蛋白質進行免疫墨點法分析後,我們發現抗體血清在第四週效價最高。 探究PLF1蛋白質在脂肪生成過程中的表現,我們發現PLF1蛋白質在脂肪細胞分化期間,隨著分化時間的增加,表現量也增加,這結果與RT-PCR分析mRNA之表現是一致的。此表現模式可完全對照在肌肉生成過程。由於PLF1是分泌性蛋白質,我們嘗試在肥胖病人開刀減重治療前後的血清中,偵測此蛋白質,惜未能所獲,可能是血中PLF1濃度太低,或是抗體的品質較差;然而在肥胖病人開刀前後的血漿中,我們意外發現在45-kDa的一個蛋白質與我們的抗體有反應,而且其表現有差異性。同樣地,在老鼠植入癌細胞及其轉移時的血清中,我們無法在24-kDa的位置偵測到PLF1,但是在相同的血清中,在45-kDa的位置也偵測到一個蛋白質。 總結,我們完成抗體的製備,在3T3-L1細胞分化成脂肪細胞的過程中,顯示PLF1蛋白質的表現量是增加的。在血清中無法偵測到PLF1蛋白質,可能是血中濃度太低,或是抗體的敏感度較差。未來,可利用抗體的專一性進行純化,以為進一步研究。 Proliferin (PLF) is a secreted glycoprotein in the prolactin-growth hormone family. PLF1 has been shown to be expressed in the fibroblasts but was suppressed during myogenic differentiation. PLF is actively involved in cell proliferation and selectively represses myogenic-specific transcription that modulates multiple muscle-specific genes. Previously, we have found that PLF1 mRNA was differentially expressed during adipocyte differentiation. To further elucidate the role of PLF1 protein in the biological system including adipogenesis, body weight regulation and cancer cell proliferation/metastasis, we aimed to express recombinant proteins, producing a polyclonal antibody, and evaluate the level of protein expression of PLF1 in various biological systems. We have cloned the full-length cDNA of the PLF1 into prokaryotic expression vector and found that the expression of the protein was predominantly insoluble and in the inclusion bodies. We have solubilized this protein and performed protein refolding to obtain active protein with success. Antibodies were obtained by immunizing rabbits via intrasplenic injection of this soluble protein. With monitoring of the titer of antibody, we found immune serum at 4th week contained a high titer of antibody as evidenced by Immunoblotting analysis on the recombinant proteins. To explore the protein expression of PLF1 during adipogenesis, we found the expression of PLF1 protein increased with adipocyte differentiation, consistent with the expression of mRNA analyses by RT-PCR. This expression pattern is quite contrast to myogenesis. Since PLF1 is a secretory protein, we tried to detect this protein in the serum of obese before and after weight reduction surgery. Unfortunately, we could not detect PLF1, possibly due to the low concentration of this protein or the quality of the antibody. However, we found a 45-kDa protein reactive to our antibody that showed difference in the plasma samples of obese subjects after surgery. Similarly, we could not detect 24-kDa PLF1 in the serum of mouse sera from the implanted cancer with and without metastasis. But, a 45-kDa protein was also detected in those samples. In conclusion, we raised the PLF1 antibodies and showed the increased protein expression of PLF1 during 3T3 adipocytes differentiation. The level of PLF1 in the serum could not be detected possibly due to low concentration of this protein or the poor sensitivity of the antibody. Further purification of the specific antibodies is warranted for future investigation. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/51368 | 其他識別: | zh-TW |
顯示於: | 分子醫學研究所 |
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