磷酰肌醇-3激酶途径与bcr/abl基因介导的白血病细胞增殖和抗凋亡信号传导

The effects of the phosphatidylinositol 3-kinase pathway on the leukemia cell's proliferation and anti-apoptosis induced by bcr/abl oncogene.

目的探讨磷酰肌醇-3激酶途径在K562、NB4和HL60细胞增殖和凋亡抗性中的不同作用。方法短期培养法直接法G显带检测K562细胞和CML患者骨髓原代细胞的染色体核型,RQ-PCR检测K562细胞和CML患者骨髓原代细胞的bcr/abl基因;用磷酰肌醇-3激酶特异抑制剂渥曼青霉素(WT)抑制磷酰肌醇-3激酶活性,经细胞生长曲线测定、半固体集落形成实验、流式细胞膜联蛋白V标记技术检测细胞凋亡百分比和凋亡指数。观察K562、NB4和HL60细胞增殖能力及凋亡抗性的变化。统计学采用t检验。结果K562细胞G显带检出Ph染色体,RQ-PCR检测K562细胞存在bcr/abl基因;与标准Ph染色体表达的CML患者骨髓原代细胞的bcr/abl基因完全吻合;K562、NB4和HL60细胞在24h、48h、72h的增殖抑制率分别为41.33%、57.46%、65.85%和26.29%、5.51%、2.10%及32.14%、17.14%、13.14%。生长曲线显示WT抑制磷酰肌醇-3激酶途径可显著抑制K562细胞的增殖(P<0.05),而对NB4和HL60细胞增殖无明显影响(P>0.05)。K562、NB4和HL60细胞加和不加WT,培养14d后的集落形成率分别为:16.15%和7.60%,5.90%和6.10%,6.60%和6.40%。集落形成抑制率为52.94%、3.39%和3.03%。WT抑制磷酰肌醇-3激酶途径可显著抑制K562细胞的集落形成(P<0.05),而对NB4和HL60细胞集落形成无明显影响(P>0.05)。K562、NB4和HL60细胞加WT,AraC,WT+AraC作用24h后的凋亡细胞百分比分别为(17.27±1.94)%、(17.00±3.36)%、(27.60±4.36)%;(13.25±4.12)%、(20.55±8.57)%、(17.56±7.97)%;(11.60±1.27)%、(22.07±5.62)%、(20.50±7.97)%。凋亡指数分别为8.91±3.86、6.88±2.66、13.39±4.49;7.79±2.75、9.35±4.19、7.61±6.35;3.03±0.56、3.79±0.93、5.27±2.69。提示渥曼青霉素抑制磷酰肌醇-3激酶途径可促进由AraC诱导下的K562细胞的凋亡(P<0.05),而对NB4和HL60细胞凋亡无明显影响(P均>0.05),NB4和HL60细胞凋亡主要受药物的细胞毒作用。结论渥曼青霉素可以通过抑制磷酰肌醇-3激酶通路抑制K562细胞的增殖,促进K562细胞的凋亡。磷酰肌醇-3激酶途径通路是bcr/abl基因介导的白血病细胞增殖和抗凋亡信号传导的重要信号传导途径。

Objective To investigate the effects of the phosphatidylinositol 3-kinase（PI3K） pathway on the leukemia cell＇s proliferation and anti-apoptosis induced by bcr/abl oncogene.Methods We determined the chromosome types of the primary cells of K562 cells and the cells in CML patients bone marrow by direct investigating of the G-banding after a short time culture, and detected the level of bcr/abl gene by RQ-PCR. We inhibited the activity of the PI3K by using Wortmannin（WT）.We counted the cell＇s clones on semisolid medium and detected the cells growth curves. We obtained the percentage of the apoptosis cells and the apoptosis index（AI）, and analysed the different potentials of proliferation and anti-apoptosis among K562 cells,NB4 cells and HL60 cells. Differences between the values were determined using Student＇s t-test. Significance was determined as P 〈 0.05.Results The K562 cells expressed bcr/abl gene.The inhibition rates of proliferation in K562 cells at 24h,48h and 72h were 41.33%,57.3% and 65.85%,while in NB4 cells were 26.29%,5.51% and 2.10%,and in HL60 cells were 32.14%,17.14%and 13.14%.We found that the proliferation of K562 cells was suppressed significantly by inhibition of the PI3K pathway（P 〈 0.05）,but there was no marked difference in NB4 cells and HL60 cells（P 〉 0.05）.The rates of clonal formation in K562 cells,NB4 cells and HL60 cells separately were 16.15%,5.90%and 6.10% after cultured for 14d,while in the control groups were 7.60%, 6.10% and 6.40% .The results suggested that the clonal formation were suppressed markedly because the PI3k was inhibited by WT（P 〈 0.05）,and there had no difference in NB4 cells and HL60cells（P 〉 0.05）. The percentages of apoptosis cells in K562 cells which cultured with WT,AraC and WT＋AraC were 17.27±1.94%,17.00±3.36% and 27.60±4.36%;13.25±4.12%,20.55±8.57% and 17.56±7.97%; and 11.60±1.27%, 22.07±5.62%, and 20.50±7.97%. The apoptotic index（AI） of these cells were 8.91±3.86,6.88±2.66 and 13.39±4.49; 7.79±2.75,9.35±4.19 and 7.61±6.35; 3.03±0.56,3.79±0.93 and 5.27±2.69. The results suggested that it promoted the apoptosis of the K562 cells induced by AraC,the PI3K pathway was inhibited by WT,at the same time there had no notable effects on the apoptosis of NB4 and HL60 cells,which were controlled by drugs cytotoxic effects.Conelusion WT can promote the apoptosis and suppress the proliferation of K562 cells throuth inhibiting the PI3k pathway.The PI3k pathway is an important transmission path in the leukemia cells＇ proliferation and anti-apoptosis induced by bcr/abl oncogene.