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 β- 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-1-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.
题目: 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.
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。.