携带TFPI-2基因的真核表达载体对SHI-1细胞生长的抑制作用

Inhibitory Effect of Eukaryotic Expression Vector Bearing TFPI-2Gene on SHI-1 Cell Growth

目的:构建人组织因子途径抑制物-2(TFPI-2)核表达载体,探讨TFPI-2基因对急性单核细胞白血病细胞株(SHI-1)生长的影响。方法:通过基因化学合成的方式得到TFPI-2的cDNA,将TFPI-2基因和线性载体片段进行连接,并插入载体PEGFP-N1多克隆位点处,再将真核表达载体PEGFP-N1-TFPI-2转染SHI-1细胞,在荧光显微镜下观察TFPI-2转染SHI-1细胞的情况;应用MTT法检测TFPI-2基因不同时间段对SHI-1细胞相对生长率的影响;RT-PCR检测TFPI-2转染前后每组细胞中TFPI-2 mRNA表达水平;采用Western blot法检测TFPI-2蛋白表达。将成功转染PEGFP-N1-TFPI-2载体的细胞命名为SHI-1-TFPI-2(实验组),转染空载体pEGFP-N1的细胞和未转染细胞设为对照组,分别命名为SHI-1-V和SHI-1-P。结果:成功构建了人TFPI-2基因真核表达载体PEGFP-N1-TFPI-2;将载体PEGFP-N1-TFPI-2转染SHI-1细胞,24 h后在荧光显微镜下可见荧光细胞,表明载体PEGFP-N1-TFPI-2成功转入SHI-1细胞;48和72 h后荧光细胞数目增多。RT-PCR结果显示:TFPI-2基因与内参β-actin条带灰度比值实验组高于对照组,灰度比值分别为:SHI-1-V组:0.51±0.04、SHI-1-P组:0.52±0.03、SHI-1-TFPI-2组:0.87±0.08;SHI-1-TFPI-2组与SHI-1-V和SHI-1-P组比较差异有统计学意义(P<0.05)。结论:PEGFP-N1-TFPI-2中TFPI-2基因的表达能抑制SHI-1细胞生长,此为未来白血病的基因治疗提供了研究方向。

Objective:To construct a eukaryotic expression vector of human tissue factor pathway inhibitor-2(TFPI-2)and to investigate the effect of TFPI-2 gene on the growth of acute monocytic leukemia cell line(SHI-1).Methods:The cDNA of TFPI-2 was obtained by genetic chemical synthesis,the TFPI-2 gene and the linear vector fragment were ligated and inserted into the multiple cloning site of PEGFP-N1 vector,and the eukaryotic expression vector PEGFP-N1-TFPI-2 was transfected SHI-1 cells,then the obtained SHI-1 cells was observed by fluorescence microscopy;MTT assay was used to detect the effect of TFPI-2 gene on the relative growth rate of SHI-1 cells at the different time-point;RT-PCR was used to detect TFPI-2 mRNA expression levels in the cells of each group before and after TFPI-2 transfection;TFPI-2 protein expression was detected by Western blot.The cells which successfully transfected with PEGFP-N1-TFPI-2 vector were named as SHI-1-TFPI-2(experimental group),and the cells transfected with the empty vector pEGFP-N1 and the untransfected cells were named as SHI-1-V and SHI-1-P and used as the control group.Results:The human TFPI-2 gene eukaryotic expression vector PEGFP-N1-TFPI-2 was successfully constructed,then the transfected into SHI-1 cells,observed by fluorescence microscopy 24 hours later,as a result,the PEGFP-N1-TFPI-2 was successfully transferred into SHI-1 cells,and the number of fluorescent cells increased after 48 h and 72 h.RT-PCR showed that the gray scale ratio of TFPI-2 gene to p-actin in the experimental group was higher than that in the control group.The gray scale ratio was 0.51±0.04 in SHI-1-V group,0.52±0.03 in SHI-l-P group,0.87±0.08 in SHI-1-TFPI-2 group,and the difference between SHI-1-TFPI-2 and SHI-1-V,SHI-1-P group was statistically significant(P<0.05).Conclusion:The expression of TFPI-2 gene in PEGFP-N1-TFPI-2 can inhibit the growth of SHI-1 cells,which provides a research direction for gene therapy of leukemia in the future.