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本研究的目的在于建立检测CML28mRNA的SYBRGreenI实时定量PCR(RQPCR)的方法,并探讨CML28表达水平与异基因造血干细胞移植(AlloHSCT)后白血病复发之间的相关性,为此构建了pcDNA3.1HisACML28质粒作为定量模板,选择14例白血病患者进行研究。14例患者包括:10例慢性髓系白血病(CML),3例急性髓系白血病(AML),1例Ph阳性急性淋巴细胞白血病(Ph+ALL)。应用RocheLightCyclerPCR仪动态监测患者移植前后不同时间段的50份骨髓单个核细胞中CML28表达水平。研究结果表明:RQPCR的灵敏度为10-4(0.05ng)水平,标准品日间差及日内差均小于10%,重复性好。初治组中AML和CML加速期(CMLAP)与急变期(CMLBC)患者CML28呈高表达(6.58±2.34)×10-2,预处理前组CML28水平为(2.19±0.32)×10-2,移植后1月组CML28水平为(1.35±1.28)×10-2,移植后3月及以后组(以下简称移植后3月组)CML28水平为(4.57±6.39)×10-3。定期随访表明,3例CMLAP或BC患者与1例CML慢性期(CMLCP)患者,在AlloHSCT3个月后检测仍呈阳性,其中2例CML28mRNA的水平<2×10-2,获无病生存;2例CML28水平>2×10-2者,一年内均出现复发,其中1例死亡,另1例行第2次AlloHSCT后CML28水平虽然迅速下降,但仍>2×10-2,2个月后再次复发。结论:CML28水平与疾病的演变过程存在良好的相关性,对于造血干细胞移植受者,动态监测CML28水平比单一的结果价值更大,实时定量检测移植后CML28水平对预测白血病复发有一定的意义。
The purpose of this study was to establish a real-time quantitative PCR (RQPCR) method for detection of CML28 mRNA and to explore the correlation between the expression level of CML28 and the relapse of leukemia after allogeneic hematopoietic stem cell transplantation (AlloHSCT). For this purpose, a pcDNA3.1HisACML28 plasmid As a quantitative template, 14 leukemia patients were selected for the study. Fourteen patients included 10 chronic myeloid leukemia (CML), 3 acute myeloid leukemia (AML) and 1 Ph positive acute lymphoblastic leukemia (Ph + ALL). The RocheLightCyclerPCR instrument was used to monitor the expression of CML28 in 50 bone marrow mononuclear cells at different time points before and after transplantation. The results showed that the sensitivity of RQPCR was 10-4 (0.05 ng), the difference between the standard and intraday difference was less than 10%, and the repeatability was good. In the untreated group, CML28 expression was significantly higher in patients with CMLAP and CMLBC (6.58 ± 2.34) × 10-2 and (2.19 ± 0.32) × 10-2 in pre-treatment group, The level of CML28 in January after transplantation was (1.35 ± 1.28) × 10-2, and the level of CML28 was (4.57 ± 6.39) × 10-3 in 3 months and after transplantation (hereinafter referred to as 3 months after transplantation). Regular follow-up showed that in 3 patients with CMLAP or BC and 1 patient with chronic phase CML (CMLCP), the test was still positive after 3 months of AlloHSCT, of which 2 patients with CML28 mRNA <2 × 10-2 had disease-free survival; 2 Cases of CML28 levels> 2 × 10-2, recurrence within a year, of which 1 died, the other after a second AlloHSCT CML28 levels decreased rapidly, but still> 2 × 10-2, 2 months later Recurrence again. CONCLUSION: There is a good correlation between the level of CML28 and the evolution of the disease. For the recipients of hematopoietic stem cell transplantation, the dynamic monitoring of CML28 level is more valuable than the single result. Real-time quantitative detection of CML28 level after transplantation has certain significance in predicting the relapse of leukemia.