Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22774
標題: 人類臍帶幹細胞中酪胺酸激酶相關之細胞生長與肌動蛋白調控蛋白相關性之探討
Involement of actin regulatory protein expression in tyrosine kinase related cell growth of undifferentiated cultured human umbilical stem cells
作者: 李瑞祐
Li, Rui-You
關鍵字: Human umbilical cord stem cell;人類臍帶幹細胞;Wharton's jelly cell;Genistein;actin polymerization / depolymerization;Formin-2;Profilin;Caldesmon;Tropomyosin.;Wharton’s jelly cell;Genistein;肌動蛋白聚合/裂解;Formin-2;Profilin;Caldesmon;Tropomyosin
出版社: 生命科學系所
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摘要: 
細胞內肌動蛋白聚合與降解的動態重組活性可調控細胞分裂以及影響細胞增殖的狀況。Formin-2(FMN-2)屬於formin同源蛋白,在小鼠以及人類神經組織細胞內專一表現的蛋白。先前研究發現:在小鼠胚體神經系統及出生後的大腦的發育階段有高度的基因表現。近期研究報告也指出,此種蛋白亦表現在小鼠卵母細胞中,此蛋白被認為於小鼠卵母細胞減數分裂系統中,可控制紡錘絲位置及染色體分離,進一步將第一極體擠壓成型。然而,目前對於FMN-2蛋白表現於其他組織中,以及調控肌動蛋白和影響細胞生長的相關機制並不清楚。人類臍帶所萃取出間葉幹細胞Wharton’s jelly cell ( WJCs ),具有自我更新、增生快速,以及可分化成多種形態細胞的潛能,例如:脂肪細胞、軟骨細胞、硬骨細胞、肌腱細胞、骨骼肌細胞、平滑肌細胞、心肌細胞等。本研究論文旨在探討FMN-2表現在WJCs內所扮演之角色。Genistein屬於酪胺酸激酶的抑制劑,能抑制EGF receptor及Src kinase等蛋白,此藥物能導致人類臍帶間葉幹細胞週期停滯於G0/G1期;相同的,運用細胞計數及MTT assay均偵測到能降低細胞增殖。因此,進一步運用共軛焦螢光顯微鏡,觀察細胞體內調控蛋白(FMN-2, profilin, tropomyosin, caldesmon, F-actin) 因Genistein降低細胞增殖是否影響調控蛋白分佈狀況。結果顯示,FMN-2、profilin均有停滯在peri-nuclear區域;另外,caldesmon及tropomyosin有向stress fiber 趨近現象,而tropomyosin isoform轉變以及分佈均有變化,此現象可能導致細胞增殖下降之主要因素。實驗結果證實:細胞內加速肌動蛋白絲聚合之蛋白(FMN-2、profilin),經由Genistein導致細胞分裂時需要束狀肌動蛋白絲無法形成,而 caldesmon、tropomyosin穩定肌動蛋白絲導致無法釋放出自由型式G-actin,此外,tropomyosin同分異構物的轉變似乎跟細胞成熟分化之機制有關,未來可對於幹細胞在細胞分裂及成熟分化時細胞骨架之變化做進一步探討。

It is generally accepted that dynamic reorganization of actin polymerization and depolymerization is the basic mechanism for maintaining cell polarization, cell proliferation and regulating cell division. In contrast to Arp2/3 which generates branched filaments, formins together with profilin nucleate straight actin filaments. Formin-2 is a formin homologous protein that is expressed in the brain, the spinal cord and in oocytes. Studies with formin gene knock out (KO) mice showed that the KO mice displayed the deficient meiosis of oocytes, suggesting the formin may control the actin filament reorganization and cell division. Physiological functions of formin-2 in the cells are not currently understood. it has not been determined if formin-2 is expressed in other tissues and how an actin regulator could control cell cycle progression in cells. In this study, we found that formin-2 mRNA and protein are expressed specifically in cultured human umbilical Wharton jelly cells based on the data obtained with a semi-quantitative RT-PCR and a western blotting analysis. These umbilical cells have been shown to have the self-renewal and differentiation capacity of mesenchymal stem cells. The expression level of forming-2 within the cultured cells is not varied by cell passages from P4 to P10. Genistein is known to be a specific inhibitor of tyrosine-specific protein kinase, ie., EGF receptor kinase, and Src. In this study, we found that this compound has growth arrested cultured human umbilical stem cells at the G0/G1 phase in cell cycle determined by the flow cytometry, as well as reduced the cell survival measured by the MTT assay and cell counting. To determine if forming-2 is involved in cell growth of the undifferentiated stem cells, we examined the effects of genistein on the cellular distributions of FMN-2, profilin, tropomyosin, caldesmon, F-actin within the cells by using a confocal fluorescence scanning microscope accompanied by applying a specific antibody against each protein. Results obtained indicated that genistein induced the translocation of actin nucleating proteins, forming-2 and profilin to the peri-nuclear areas. Caldesmon and tropomoysin become associated with the stress fiber. It seems likely that the actin filament remodeling in the peri-nuclear area and some binding proteins associated with the stress fiber might have the functions for inhibition of cell proliferation in undifferentiated stem cells. Future study might be invested to study further if changes in cell cytoskeleton remodeling are also associated with differentiation of mesenchymal stem cells.
URI: http://hdl.handle.net/11455/22774
其他識別: U0005-2508200819320900
Appears in Collections:生命科學系所

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