论文部分内容阅读
目的构建共表达绿色荧光蛋白(green fluorescent protein,GFP)基因和人磷酸葡萄糖异构酶(phosphoglucose isomerase,PGI)基因的慢病毒载体,初步探讨干扰人PGI基因对白血病细胞增殖的影响。方法 RT-PCR、Westernblot检测人单核细胞、K562、KG1-α、HL-60细胞中PGI mRNA和蛋白表达水平,筛选高表达PGI的白血病细胞系;构建人PGI基因siRNA的慢病毒载体及阴性对照,经293T细胞包装后,获得可表达人PGI基因siRNA的慢病毒载体及阴性对照;分别使用重组病毒(干扰组)、原始病毒(阴性对照组)和等量PBS(空白对照组)转染白血病KG1-α细胞,筛选稳定转染细胞株。RT-PCR和Western blot检测干扰组、阴性对照组及空白对照组KG1-α细胞PGI基因表达水平;CCK-8检测各组细胞增殖情况并作生长曲线。结果 PGI基因在白血病KG1-a细胞中高表达;成功构建了稳定低表达PGI的白血病细胞株(KG1-α-siPGI);低表达PGI基因的KG1-α细胞增殖能力较空白对照组明显降低,差异具有统计学意义(P<0.05)。结论成功构建了低表达PGI基因的白血病KG1-α细胞株,干扰PGI基因的表达能够抑制KG1-α细胞的生长。
Objective To construct a lentiviral vector coexpressing green fluorescent protein (GFP) gene and phosphoglucose isomerase (PGI) gene and to investigate the effect of interfering human PGI gene on the proliferation of leukemia cells. Methods The expression of PGI mRNA and protein in human monocytes, K562, KG1-α and HL-60 cells was detected by RT-PCR and Western blot. The leukemic cell lines with high PGI expression were screened. Control, 293T cells were packaged, get lentiviral vector can express human PGI gene siRNA and negative control; respectively, using recombinant virus (interference group), the original virus (negative control group) and an equal volume of PBS (blank control group) transfected Leukemia KG1-α cells, screening stable transfected cell lines. The expression of PGI gene in KG1-α cells in the interference group, the negative control group and the blank control group was detected by RT-PCR and Western blot. The proliferation of each group was detected by CCK-8. Results PGI gene was overexpressed in leukemic KG1-a cells. KG1-α-siPGI was successfully constructed with low expression of PGI gene. The proliferation of KG1-α cells with low expression of PGI gene was significantly lower than that of the control group Statistically significant (P <0.05). Conclusion KG1-α leukemia cell line with low expression of PGI gene was successfully constructed and the expression of PGI gene was inhibited. The growth of KG1-α cells was inhibited.