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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 the advantages of Western blotting and flow cytometry (FCM) in the detection of intracellular translational tumor protein (TCTP) and to analyze their application value in clinical detection. METHODS: NB4 and NB4-R1 cells were treated with 1 μmol/L all-trans-retinoic acid (ATRA) for 0-96 h. The changes of TCTP protein in cells were detected by Western blotting and FCM. The Bland-Altman analysis was used to analyze the consistency of the test results. FCM was used to detect the level of TCTP protein in primary bone marrow samples from patients with acute leukemia. Results: The results of Western blotting showed that TCTP in NB4 cells decreased after ATRA treatment. The relative values of TCTP and β-actin gray ratio at each time point (0, 24, 48, 72, and 96 h) were 1.00 and 0.97, respectively. ± 0.05, 0.91 ± 0.03, 0.85 ± 0.05, and 0.72 ± 0.02, but remained unchanged in NB4-R1 cells. The results of FCM showed that the positive rates of TCTP in NB4 cells after treatment with ATRA were (98.93±0.49)%, (98.83±0.65)%, (96.77±1.45)%, (90.93±1.35)%, and ( 72.17±5.57)% remained unchanged in NB4-R1 cells. According to the Bland-Altman method, the consistency interval limit for the 95% measurement of the two methods was calculated to be (-0.081, 0.111). Twenty-nine of the 30 groups of data measured in this study fell within the range. The detection of TCTP levels in myeloid cells from 11 patients with acute leukemia and 9 patients with non-leukemia showed that the expression of TCTP in myeloid cells of non-leukemia patients was low, while that of patients with leukemia was relatively high. The difference between the two was statistically significant. (P<0.01). Conclusion: There is a good agreement between Western blotting and the results of FCM detection of intracellular TCTP protein levels, whereas FCM is more suitable for the detection of primary patient specimens. The level of intracellular TCTP may be related to the occurrence and development of leukemia, and may be used as a molecular marker for clinical leukemia detection in the future.