应用GFP质粒构建及转染技术探讨Syk(L)核移位机制

【目的】通过研究Syk(L)在乳腺癌细胞亚细胞器中的分布和移位机制,探讨Syk(L)抑制乳腺癌的可能性机制。【方法】采用细胞胞核、胞浆分离技术和Westernblot的方法,检测乳腺癌细胞系BT474中两种Syk蛋白异构体在细胞内的分布。构建绿色荧光蛋白(GFP)和Syk(L)核移位相关功能域融合蛋白质粒,转染入Cos7细胞内,荧光显微镜下观察融合蛋白在细胞内的分布。【结果】BT474细胞的细胞浆中可检测到Syk(S)和Syk(L)的表达,细胞核中只检测到Syk(L)的表达。在Cos7细胞中,GFP与Syk(L)的缺失域(deletiondomain,DEL域)融合蛋白均匀分布于细胞内,GFP与Syk(L)的IDB域融合蛋白聚集于细胞核中;将DEL域上的294K、300K、及305K突变域非碱性氨基酸后构建成GFP-IDB(L)-M,突变的融合蛋白则失去核聚集现象。【结论】在乳腺癌细胞中,Syk(L)可从细胞胞浆移位到细胞核内。Syk(L)的IDB域具有完整的核移位功能,能将融合的胞浆蛋白GFP转移到细胞核内;DEL域的碱性氨基酸具有核定位信号功能。Syk(L)在乳腺癌细胞中的抑癌功能与其核移位有关。

Using GFP Constructs Making and Transfection Technology to Explore Mechanism of Syk(L) Nuclear Translocation

Objective By studying the subcellular localization of Syk(L)in breast cancer cells and the mechanism of its nuclear translocation,explore the possible mechanism of Syk(L)tumor suppression function.Methods Cytosolic and nuclear fractionation experiments were used to separate the cytosolic and nuclear cell lysate of breast cancer cell lines BT474.Western blot was used to analyze the subcellular localization of Syk.Green fluoresent protein(GFP)and Syk(L)nuclear translocation domain fusion proteins were constructed and transfected into Cos 7 cells.The subcellular localization of fusion proteins were observed under fluorescence microscope.Results Both Syk(S)and Syk(L)was found in the cytosolic extracts of breast cancer cell BT474,while only Syk(L)was found in the nuclear extracts.GFP and Syk(L)'s deletion(DEL)domain fusion protein was observed expressing in the cells evenly.Fused IDB domain of Syk(L)was able to transport GFP into nucleus and congregated in the nuecleus.When the basic amino acids 294K,300K and 305K in the DEL domain were mutated to non-basic amino acids asparagine,the mutated GFP-IDB(L)fusion protein lost the ability of nuclear congregation.Conclusions Syk(L)is able to translocate to nucleus from cytoplasm in breast cancer cell.The IDB domain of Syk(L)has complete nuclear translocalization function,which is able to transport cytoplasm protein GFP into nucleus.The basic amino acids in DEL domain of Syk(L)contains nuclear localization signal.Syk(L)tumor suppression function is possiblely associated with its nuclear translocation.