过表达DACT1通过调节凋亡相关蛋白对人白血病细胞增殖的抑制作用研究

Over-expression of DACT1 inhibits proliferation of human leukemic cells by regulating apoptosis related proteins

目的研究过表达DACT1通过调节凋亡相关蛋白对人白血病细胞增殖的抑制作用。方法在人白血病K562细胞中,通过基因转染试剂对空载质粒vector与DACT1质粒分别进行转染,分别作为阴性对照组与观察组,并使DACT1呈现过表达。另设置一组未经任何处理的细胞为空白对照组。通过流式细胞术、CCK-8法对DACT1过表达后K562细胞周期、增殖、凋亡的影响进行检测。并采用Western blot法对DACT1过表达后调节凋亡相关蛋白的表达进行检测。结果观察组克隆形成率为(6. 33±0. 98)%,较空白对照组(20. 74±3. 12)%、阴性对照组(20. 43±3. 32)%均明显降低(P <0. 05)。细胞增殖实验结果发现,观察组细胞增殖活性较空白对照组、阴性对照组均显著降低。观察组细胞凋亡率为(28. 97±3. 04)%,较空白对照组(0. 85±0. 12)%、阴性对照组(0. 83±0. 09)%均显著升高(P <0. 05)。观察组细胞G0/G1期比例较空白对照组、阴性对照组显著增加(P <0. 05)。观察组半胱氨酸天冬氨酸蛋白酶-3(caspase-3)、半胱氨酸天冬氨酸蛋白酶-9(caspase-9)、Bcl-2相关X蛋白(Bax)等促凋亡蛋白表达较空白对照组和阴性对照组均显著上调,而B淋巴细胞癌-2(Bcl-2)等抗凋亡蛋白的表达较空白对照组和阴性对照组均显著降低。结论在人白血病K562细胞中,DACT1过表达可阻滞细胞增殖,促使细胞凋亡,亦会影响细胞周期,使得细胞出现G0/G1期阻滞。分析其原因,可能与DACT1过表达具有潜在的抗肿瘤作用,其作用机制可能与上调促凋亡蛋白(caspase-3、caspase-9、Bcl-2)表达和抑制抗凋亡蛋白(Bcl-2)表达密切相关。

Objective To study the inhibitory effect of over-expression of DACT1 on the proliferation of human leukemia cells through regulating apoptosis related proteins. Methods In human leukemia K562 cells,gene transfection reagent( Attractene Transfection Reagent) were used to transfect the empty plasmid vector and DACT1 plasmid respectively as the negative control group and the observation group,and the DACT1 was over-expressed. Another set of cells without any treatment was blank control group. The cell cycle,proliferation and apoptosis of K562 after overexpression of DACT1 were detected by flow cytometry and CCK-8. Western blot was used to detect the expression of apoptosis related proteins after over expression of DACT1. Results The colony formation rate in the observation group was( 6. 33 ± 0. 98)%,which was significantly decreased in comparison of the blank control group( 20. 74 ± 3. 12)% and the negative control group( 20. 43 ± 3. 32)%( P < 0. 05).Cell proliferation assay showed that the cell proliferation activity in the observation group was significantly decreased in comparison of the blank control group and the negative control group. The apoptotic rate in the observation group was( 28. 97 ± 3. 04)%,which was significantly increased in comparison of the blank control group( 0. 85 ± 0. 12)% and the negative control group( 0. 83 ± 0. 09)%( P < 0. 05). The proportion of G0/G1 phase in the observation group was significantly increased in comparison of the blank control group and negative control group( P < 0. 05).The expression of cysteine aspartic protease-3( caspase-3),cysteine aspartic proteinase-9( caspase-9) and Bcl-2 related X protein( Bax) in the observation group were significantly up-regulated than those in the blank control group and the negative control group,while the expression of B-cell lymphoma-2( Bcl-2) as anti-apoptotic proteins was significantly lower than that in the blank control group and the negative control group. Conclusion In human leukemia K562 cells,over-expression of DACT1 can block new proliferation,induce cell apoptosis,and affect cell cycle,causing G0/G1 block in cells. The analysis of its causes may have potential antitumor effects with DACT1 over-expression,and its mechanism may be closely related to the expression of apoptotic protein( such as caspase-3,caspase-9,Bcl-2) and the inhibition of the expression of anti apoptotic protein( such as Bcl-2).