白血病细胞中翻译控制肿瘤蛋白的检测及其方法学比较

Detection of translationally controlled tumor protein in leukemia cells and comparison of detection methodology

目的 ：比较蛋白印迹法和流式细胞术（flow cytometry,FCM）在检测细胞内翻译控制肿瘤蛋白（translationally controlled tumor protein,TCTP）中的优势,并分析两者在临床检测中的应用价值。方法 ：用1μmol/L全反式维A酸（all-trans-retinoic acid,ATRA）处理NB4和NB4-R1细胞0~96 h,分别采用蛋白印迹法和FCM检测细胞内TCTP蛋白的变化情况,用Bland-Altman分析法分析检测结果的一致性。用FCM检测部分急性白血病患者骨髓原代标本中TCTP蛋白水平。结果 ：蛋白印迹法检测结果显示,经ATRA处理后NB4细胞内TCTP下降,各时间点（0、24、48、72、96 h）TCTP与β-actin灰度比的相对值分别为1.00、0.97±0.05、0.91±0.03、0.85±0.05和0.72±0.02,而NB4-R1细胞中则保持不变。FCM检测结果显示,经ATRA处理后NB4细胞内各时间点TCTP的阳性率分别为（98.93±0.49）%、（98.83±0.65）%、（96.77±1.45）%、（90.93±1.35）%和（72.17±5.57）%,NB4-R1细胞中保持不变。根据Bland-Altman法,计算可得2种方法测量值95%的一致性区间界限为（-0.081,0.111）,本研究所测得的30组数据中有29组落在区间内。对11例急性白血病患者和9例非白血病患者骨髓细胞内TCTP水平的检测结果显示,非白血病患者骨髓细胞内TCTP的表达较低,而白血病患者则相对较高,两者间差异有统计学意义（P〈0.01）。结论：蛋白印迹法与FCM检测细胞内TCTP蛋白水平的结果间有良好的一致性,而FCM更加适用于患者的原代标本检测。细胞内TCTP水平的高低可能与白血病的发生、发展有关,今后有可能作为临床白血病检测的分子标志物。

Objective： To compare Western blotting and flow cytometry（FCM） for detecting intracellular level of translationally controlled tumor protein（TCTP） and to analyze the value of these two methods for clinical application.Methods： NB4 and NB4-R1 cells were treated with 1μmol/L ATRA for 0-96 hours. Western blotting and FCM were used to test the changes of TCTP levels in these two cells.Bland-Altman plots were performed to analyze the agreement of these two test results. FCM technique was employed to detect the TCTP levels in primary bone marrow cells from 11 patients with acute leukemia. Results： Western blotting showed that the TCTP level was obviously declined in NB4 cells after ATRA treatment, the relative ratio of gray scale of TCTP and β-actin in ATRA-treated NB4 cells at each time point were1.00, 0.97±0.05, 0.91±0.03,0.85±0.05 and 0.72±0.02, respectively. Meanwhile, the results of FCM showed that the percentages of TCTP positive cells were（98.93 ±0.49）%,（98.83 ±0.65）%,（96.77 ±1.45）%,（90.93 ±1.35）% and（72.17 ±5.57）%, respectively. No significant difference in TCTP level was observed in ATRA-treated NB4-R1 cells by these two methods. According to Bland-Altman plots, the 95% limits of agreement of the two test results ranged from-0.081 to 0.111. In this study, there were 29 sets of data falling within the abovementioned range limits within 30 sets of measured data. TCTP levels in primary cells from leukemia patients were significantly higher than those from non-leukemia patients. Conclusions：Detection results between Western blotting and FCM are in good consistency. And FCM analysis is more suitable for detec-tion of primary cell samples than Western blotting. Moreover, cellular TCTP level may be related to genesis and development of leukemogenesis and could be served as a potential molecular marker for diagnosis of leukemia in clinical practice.