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ABL SH3-T79Y突变体联合伊马替尼对慢性粒细胞白血病细胞增殖的影响
作者:文良雪 刘鑫 李会 黄宁姝 黄峥兰 冯文莉 
单位:400016 重庆 重庆医科大学检验医学院临床检验诊断学教育部重点实验室(文良雪、刘鑫、李会、黄宁姝、黄峥兰、冯文莉) 
关键词:细胞增殖 白血病 髓系 慢性 BCR-ABL阳性 RIN1蛋白 K562/G01细胞 KCL22细胞 
分类号:R733.72
出版年,卷(期):页码:2015,40(8):616-621
摘要:

目的 分析慢性粒细胞白血病(CML) BCR-ABL蛋白的SH3结构域突变体(ABL SH3-T79Y)联合伊马替尼(IM)在体内外对CML细胞增殖的影响,并初步探讨其发挥作用的机制。方法 构建重组腺病毒载体SH3-T79Y突变体,将其联合IM处理K562/G01细胞后,采用克隆形成实验检测细胞克隆形成率,流式细胞术检测细胞周期,分析K562/ G01细胞体外增殖能力的变化。SH3-T79Y联合IM处理KCL22细胞,构建BALB/c裸鼠皮下实体瘤模型,计算皮下瘤形成率,摘取瘤体进行病理学检查,分析KCL22细胞体内增殖能力的变化。SH3-T79Y联合IM处理K562/G01细胞后,采用Western blotting检测p-BCR-ABLBCR-ABLp-CrkLCrkLCyclin D1蛋白的表达水平,分析联合作用影响CML细胞增殖的机制。实验中以PBS、腺病毒空载体及IM单独处理细胞作为对照。结果 相较于三个对照组,SH3-T79Y联合IM处理K562/G01细胞后,细胞克隆形成率(28.74%±6.64%)明显降低(P0.05),细胞周期阻滞于S期。联合处理KCL22细胞后,KCL22-BALB/c裸鼠皮下实体瘤模型成瘤率为16.7%,明显低于IM单独处理组(33.3%)PBS对照组(100%),瘤体病理学检查见大量增殖的瘤细胞;Western blotting检测结果显示联合处理后K562/G01细胞p-BCR-ABLp-CrkLBCRABLCrkLCyclin D1的表达均降低。结论 SH3-T79Y联合IM通过抑制BCR-ABLCrkL磷酸化和降低Cyclin D1表达,在体内、体外发挥了抑制CML细胞增殖的效应。

Objective To analyze the influence of SH3 domain mutant (ABL SH3-T79Y) in BCR-ABL protein of chronic myeloid leukemia (CML) in combination with imatinib (IM) on the proliferation of CML cells in vivoand vitro, and to discuss the mechanism thereof. Methods Recombinant ABL SH3-T79Y mutant adenovirus vectors which were successfully constructed in previous work was used with IM to treat K562/G01 cells, then the cell-colony forming ability of K562/G01 cells was determined by clone formation assay, and cell cycle was assessed by flow cytometry. KCL22 cells were treated by recombinant SH3-T79Y and IM to construct subcutaneous solid tumor model in Balb/c nude mice, then the formation rate of subcutaneous tumor was estimated, the pathological examination was conducted, and the proliferation ability of KCL22 cells was assayed. K562/G01 cells were treated by SH3-T79Y and IM in combination, and the expression levels of p-BCR-ABL, BCR-ABL, p-CrkL, CrkL and Cyclin-D1 protein were determined by Western blotting. Cells treated with PBS, null recombinant adenovirus vectors or IM alone served as control groups. Results Compared to the 3 control groups, clone forming rate of K562/G01 cells decreased significantly (P<0.05) and cell cycles were arrested at S phase after being combined SH3-T79Y and IM treatment. The subcutaneous solid tumor formation rate in KCL22Balb/c nude mice was 16.7% after combined SH3-T79Y and IM treatment, and large number of tumor cells were observed in tumor pathology examination. Western blotting revealed that the expression levels of p-BCR-ABL, p-CrkL, BCR-ABL, CrkL and Cyclin-D1 were decreased in K562/G01 cells. Conclusion Combined treatment of SH3-T79Y and imatinib may inhibit the proliferation of CML cells in vivoand in vitroby decreasing BCR-ABL and CrkL phosphorylation as well as Cyclin-D1 protein.

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