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目的:探讨水通道蛋白1(aquaporin-1,AQP1)过表达对人慢性髓细胞白血病(chronic myeloid leukemia,CML)K562细胞红系分化和增殖的影响。方法:以人脑cDNA文库为模板,通过PCR扩增出AQP1基因的编码序列,构建pBABE-puro-AQP1真核表达载体;感染K562细胞,筛选建立稳定过表达AQP1基因的K562细胞株(命名为K562-AQP1);实时荧光定量PCR法、细胞免疫荧光染色法及蛋白质印迹法分别检测AQP1转录和蛋白表达水平。通过MTT法检测细胞生长增殖、实时荧光定量PCR法检测γ珠蛋白表达和分光光度法检测血红蛋白含量,研究AQP1过表达对K562细胞红系分化和增殖的影响。结果:与空载体对照组相比,pBABE-puro-AQP1转染入K562细胞后AQP1 mRNA和蛋白表达水平皆有显著升高(P<0.01),K562-AQP1细胞中红系分化指标γ珠蛋白和血红蛋白表达水平明显增加,同时细胞生长速度明显降低(P<0.05)。结论:AQP1过表达可以显著促进K562细胞向红系分化,同时抑制细胞增殖。推测AQP1可能成为临床诱导分化治疗CML的基因靶点之一。
Objective: To investigate the effect of aquaporin-1 (AQP1) overexpression on erythroid differentiation and proliferation in human chronic myeloid leukemia (KML) K562 cells. Methods: The coding sequence of AQP1 gene was amplified by PCR from human brain cDNA library, and then the eukaryotic expression vector pBABE-puro-AQP1 was constructed. K562 cells were infected with K562 cells, and the K562 cell line stably overexpressing AQP1 K562-AQP1); AQP1 transcription and protein expression were detected by real-time fluorescence quantitative PCR, immunofluorescence staining and Western blot respectively. The proliferation and proliferation of K562 cells were detected by MTT assay. The expression of hemoglobin was detected by real-time fluorescence quantitative PCR and spectrophotometry. The effect of AQP1 overexpression on erythroid differentiation and proliferation was investigated. Results: The mRNA and protein expression of AQP1 in K562 cells transfected with pBABE-puro-AQP1 were significantly increased (P <0.01) compared with the control group. The erythroid differentiation index γ-globin And hemoglobin expression increased significantly, while the cell growth rate was significantly lower (P <0.05). Conclusion: Overexpression of AQP1 can significantly promote the differentiation of K562 cells to erythroid cells and inhibit cell proliferation. Speculated that AQP1 may be clinically induced differentiation of CML one of the genetic targets.