过表达SHP-1对人慢性髓系白血病急变细胞系K562生物学特征的影响

Effects of SHP-1 Overexpression on Biological Characteristics of K562 Cells

目的探讨SHP-1基因对K562细胞系增殖、凋亡、细胞周期分布和集落形成的影响。方法将携带SHP-1基因和绿色荧光蛋白(EGFP)基因的慢病毒表达载体转染K562细胞,SYBR Green荧光定量PCR和Western blot法检测转染细胞SHP-1 m RNA和蛋白的表达情况;CCK-8法检测细胞增殖;流式细胞术检测细胞凋亡率和细胞周期分布;光学显微镜和电子显微镜下观察细胞形态;TUNEL法检测细胞凋亡;培养细胞集落并比较不同组间集落形成的数量和大小。结果培养第3天,K562SHP-1细胞的OD值为(0.35±0.02),K562EGFP细胞的OD值为(0.47±0.05),两者差异有统计学意义(P=0.011)。随着培养天数的延长,与K562EGFP细胞相比K562SHP-1细胞出现G0/G1期细胞比例增加和G2/M+S期细胞比例降低及凋亡增加,培养第5天K562SHP-1细胞G0/G1期比例为(56.37±2.27)、凋亡率为(26.13±1.16),K562EGFP细胞G0/G1期比例为(31.67±3.21),凋亡率为(9.00±1.22),两者比较差异有统计学意义(P=0.000)。K562SHP-1细胞集落形成能力(26.3±5.2)较K562EGFP细胞(54.7±8.6)有所降低(P=0.000);K562SHP-1细胞BCR-ABL1蛋白的表达水平(0.78±0.15)低于K562EGFP细胞(1.27±0.24)(P=0.040)。结论过表达SHP-1基因抑制K562细胞增殖和集落形成,促进细胞凋亡,导致细胞周期阻滞在G0/G1期,并降低K562细胞中BCR-ABL1蛋白水平。

Objective To investigate the effects of SHP-1 gene on the proliferation, apoptosis, cell cycle, and colony formation of K562 cells line in vitro. Methods K562 cells were infected with the lentiviral plasmids carrying the specified retroviral vector(p EX-SHP-1-puro-Lv105) or the control vector(p EX-EGFPpuro-Lv105). The expression of SHP-1 m RNA and protein were determined by SYBR Green-based q RTPCR and Western blot. The proliferation of cells was detected by CCK-8 assay, and the apoptosis rate and cell cycle were assessed by flow cytometry. Besides, cell morphology was observed by light and electron microscopy. Cell apoptosis was detected by TUNEL method. Cell colonies were cultured and the number of colonies was compared between groups. Results CCK-8 assays showed that the cell proliferation rate was sharply reduced in K562SHP-1 cells(0.35±0.02), compared with K562 EGFP cells(0.47±0.05) at the third day of culture(P=0.011). Flow cytometry analysis showed that K562SHP-1 cells were arrested in G0/G1 phase(56.37±2.27), compared with K562 EGFP cells(31.67±3.21)(P=0.000) at the fifth day of culture; and the percentage of apoptotic K562SHP-1 cells(26.13±1.16) was significantly higher than that of apoptotic K562 EGFP cells(9.00±1.22)(P=0.000). The colony forming ability of K562SHP-1 cells(26.3±5.2) was decreased compared with K562 EGFP cells(54.7±8.6),(P=0.000). SHP-1 overexpression caused a slight decrease of BCR-ABL1 protein in K562SHP-1 cells compared with K562 EGFP cells(P=0.040). Conclusion The overexpression of SHP-1 gene could inhibit the proliferation and colony formation of K562 cells, induce cell apoptosis, result in cell cycle arrest at G0/G1 phase, and decrease the level of BCR-ABL1 protein.