实时定量RT-PCR监测造血干细胞移植前后CML28mRNA表达  

Monitoring CML28 mRNA Levels in Patients Before and After HSCT by Real-time Quantitative RT-PCR

作　　者：张东华[1] 戴敏[1] 周红升[1] 汪涯雅[1] 张路[1] 张丽[1] 王斌[2] 曹文静[2] 

机构地区：[1]华中科技大学同济医学院附属同济医院血液科 [2]华中科技大学同济医学院附属同济医院检验科,武汉430030

出　　处：《中国实验血液学杂志》2005年第5期843-847,共5页Journal of Experimental Hematology

基　　金：湖北省卫生厅资助项目(编号:JX1B047)

摘　　要：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。Abstract The purpose of this study was to establish a SYBR Green I real-time quantitative RT-PCR method for investigating the correlation between CML28 mRNA expression levels and relapse of leukemia after allo-hematopoietic stem cell transplantation （HSCT）, pcDNA3, 1HisA-CML28 plasmid had been constructed as the standard template, Serial monitorting of CML28 mRNA levels by SYBR Green I real-time quantitative RT-PCR technique was performed in 14 patients, including 10 patients with CML and 3 patients with AML,1 patient with Ph^＋ ALL. The results showed that the sensitivity of the established method was at 10^-4（ 0, 05 ng） level, with interassay variation and intraassay variation of standard samples both below 10%, The CML28 was highly expressed in AML and CML-BP or AP. CML28 level in newly diagnosed group was （6, 58 ± 2, 34）× 10^-2 in pre-conditioning regimen group was （2. 19 ± 0, 32 ）× 10^-2, in group that l month after HSCT was（ 1.35 ± 1,28 ）× 10^-2, in group that 3 months after HSCT was （4.57 ± 6. 39）× 10^-3, CML28 can be detected 3 months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BC. 2 of them with low level（ 〈 2 ×10^-2） survived without relapse, the other 2 case with high level（ 〉 2 ×10^-2） relapsed within one year, l case died and 1 case received the second time HSCT, CML28 level decreased rapidly after HSCT, but still higher than 2 ×10^-2 and relapse has taken place, The conclusions was made that CML28 mRNA level is obviously correlated with the development of leukemia, Serial quantification of CML28 mRNA levels are necessary for HSCT recipients, and more informative than a single detection. Using of this assay to evaluate MRD in the patients received HSCT is helpful for predicition of relapse.

关 键 词：白血病 ALLO-HSCT CML28 实时定量RT-PCR SYBR Green Ⅰ 

分 类 号：R733.72[医药卫生—肿瘤] R457.7[医药卫生—临床医学]