人B细胞淋巴瘤/白血病-2基因质粒体的构建和表达及B细胞淋巴瘤/白血病-2基因在U251细胞中的作用

Construction of plasmid of B lymphocytes/leukemia-2 and its expression and function in U251 cells

目的 构建人B细胞淋巴瘤/白血病-2（bcl-2）基因质粒体并检测其在胶质瘤细胞株U251内的表达.方法 构建PEGFP-bcl-2质粒体,应用FUGENE HD转染质粒体进入U251,G418筛选,挑出单克隆扩增,建立稳定细胞株N1-U251和bcl-2-U251.将实验分为：空白组（U251细胞）、N1组（N1-U251）、bcl-2组（bcl-2-U251）,检测bcl-2基因和蛋白表达；使用噻唑蓝（MTT）法检测N1组（N1-U251）,bcl-2组细胞活性.结果 PEGFP-bcl-2质粒体构建成功,N1-U251、bcl-2-U251稳定细胞株构建成功,在空白组、N1组、bcl-2组bcl-2蛋白表达量分别为：0.012±0.003、0.011 ±0.003、0.115±0.025.细胞活性分别为：1.015±0.005、1.120±0.004、3.312±0.004,bcl-2组与对照组比较差异有统计学意义（P＜0.05）.结论 成功构建人bcl-2质粒体并建立高表达bcl-2的细胞株,高表达bcl-2可以提高U251细胞活性.

Objective To construct the plasmid of B lymphocytes/leukemia-2 （bcl-2） and observe its expression in U251 cells.Methods Plasmid of bcl-2 was constructed and transfected into U251 cells by FUGENE HD.G418 was used to kill the cells that were transfected unsucessfully.Western blotting and reverse transcriptase polymerase chain reaction （RT-PCR） were used to observe the expression of bcl-2.Three groups were set up：control group,N1 group and bcl-2 group.Methyl thiazol tetrazolium （MTT） assay was used to measure the viability of U251 cells in N1 group and bcl-2 group.Results The plasmid PEGFPbcl-2 and bcl-2-U251 cells were constructed successfully.The expression of bcl-2 in control group,N1 group and bcl-2 group was 0.012 ±0.003,0.011 ±0.003 and 0.115 ±0.025,and the viability of U251 cells was 1.015 ±0.005,1.120 ± 0.004 and 3.312 ± 0.004,respectively.There was significant difference between bcl-2 group and control group （P 〈 0.05）.Conclusion The plasmid PEGFP-bcl-2 was constructed successfully and the overexpression system is also established,and bcl-2 overexpression can increase the viability of U251 cells.