LEF-1截短型基因真核荧光表达载体的构建及其在SW480细胞系中的功能研究

Functional study of the eukaryotic fluorescent expression vector of truncated lymphoid enhancer-binding factor 1(LEF-1) gene in SW480 cells

目的构建LEF-1截短型基因的真核表达荧光质粒,在真核细胞中表达,并检测其对人结肠癌细胞系SW480增殖和凋亡的影响。方法利用PCR方法从人淋巴结cDNA文库中克隆LEF-1截短型的编码基因,插入到真核表达载体pcDNA3.1中,同时插入红色荧光示踪片段mRFP。用脂质体将重组质粒pcDNA3.1-LEF-1-mRFP瞬时转染Hela细胞,通过Westernblot和流式细胞仪检测LEF-1截短型的表达,同法瞬时转染SW480细胞,观察细胞形态变化,MTT检测对细胞生长的影响,CSFE染色检测细胞的增殖情况,AnnexinV方法检测细胞凋亡情况,PI染色检测细胞周期。结果克隆LEF-1截短型的编码基因,经测序比对证实与GenBank中给出的序列一致。以含有LEF-1截短型的编码基因的真核表达荧光载体瞬时转染Hela细胞,LEF-1截短型基因的表达产物通过Westernblot和流式细胞仪方法得到证实。转染SW480细胞,光镜下观察发现部分细胞中出现颗粒,失去正常形态,细胞数目明显减少;MTT检测显示细胞活性下降,流式检测显示细胞增殖受到抑制,凋亡水平增加,细胞在G0/1期发生阻滞。结论成功构建LEF-1截短型的编码基因的真核荧光表达载体,证实其在真核细胞Hela中可以表达。同时可以抑制SW480细胞的活性和增殖,促进其凋亡,细胞被阻滞在G0/1期。

Abstract： Objective To construct a eukaryotic fluorescent expression vector of truncated lymphoid enhancer-binding factor 1 （LEF-1） gene and investigate its effect on the proliferation and apoptosis of human colonic carcinoma cell line SW480. Methods Truncated LEF-1 gene was obtained by PCR and DNA recombination from human lymphoid node cDNA library. The PCR product ofLEF-1 gene was inserted into the plasmid pMD-18T and sequenced. The truncated LEF-I gene was inserted into the eukaryotic expression plasmid pcDNA3.1 and fused with mRFP for tracing. Using LipofectamineTM 2000, the plasmid pcDNA3.1-LEF-I-mRFP was transfected into Hela cells and detected by Western blotting and fluorescence activated cell sorting （FACS）. The changes in the growth, proliferation and apoptosis of the SW480 cells were observed after transfection with the plasmids. Results The truncated LEF- 1 gene was successfully cloned. After transfection with the plasmid pcDNA3.1-LEF-I-mRFP, the Hela cells expressed the product of LEF-1 as detected by Western blotting and FACS. The growth and proliferation of SW480 cells was inhibited arid the cell apoptosis increased after transfection with the plasmid pcDNA3.1-LEF-1-mRFP, which also caused cell cycle arrest in G0/1 phase. Conclusion The eukaryotic expression fluorescent vector pcDNA3.1-LEF-1-mRFP has been constructed and expressed in eukaryotic cell line successfully. The truncated LEF-1 protein expressed in the transfected SW480 cells results in inhibition of the cell growth and proliferation with increased cell apoptosis and cell cycle arrest in G0/1 phase.