荧光定量PCR检测大剂量甲氨喋呤化疗急性淋巴细胞白血病患儿肠道乳酸杆菌和大肠埃希菌量的变化  

Study on intestinal bacterial flora by fluorescent quantitative PCR in children with acute lymphoblastic leukemia of high-dose methotrexate therapy

作　　者：段晶[1] 刘华[2] 杨武[1] 黄永坤[1] 张瑛[1] 刘梅[1] 鲁萍[1] 李海林[1] 

机构地区：[1]昆明医学院第一附属医院儿科,云南昆明650032 [2]昆明医学院第一附属医院检验科基因研究室,云南昆明650032

出　　处：《中国微生态学杂志》2008年第3期197-202,共6页Chinese Journal of Microecology

基　　金：云南省中青年学术技术带头人后备人才培养计划资助项目(2006py01-18)

摘　　要：目的研究大剂量甲氨喋呤(HDMTX)化疗急性淋巴细胞白血病(ALL)患儿肠道菌群的变化,并和健康儿童进行比较。从微生态的角度分析HDMTX化疗与肠道菌群变化的相互关系,为微生态制剂用于HDMTX化疗副作用的预防和治疗提供依据。并探讨荧光定量PCR技术在该领域应用的实用性和可行性,以便推广应用。方法收集36例HDMTX化疗前后ALL患儿及36例健康对照组儿童的粪便标本,并提取目标细菌DNA,用分子生物学专用分光光度仪进行DNA的A值测量。用细菌的16S rRNA/DNA序列设计乳酸杆菌和大肠埃希菌的引物,行常规PCR完成细菌的定性,然后取准确定量的两种细菌DNA,经系列稀释后做荧光定量PCR,制作出标准曲线,待测样品同时进行荧光定量PCR反应,并和标准曲线进行比较,获得各样品中两种细菌的量。结果HDMTX化疗ALL患儿的肠道菌群与健康儿童比较发生了明显的变化,患儿化疗前1天、化疗后第3天、化疗后第7天和对照组儿童的粪便标本中细菌的A260值分别为(2436.3±768.6)、(1496.5±577.1)、(1966.6±598.3)和(3479.3±870.5)ng/μl;患儿化疗前1天、化疗后第3天、化疗后第7天和对照组的肠道乳酸杆菌数量的对数值分别为(8.10±0.43)、(6.73±0.45)、(7.45±0.43)和(9.12±0.50)ng/μl,而大肠埃希菌数量的对数值分别是(6.62±0.4)、(5.96±0.42)、(7.02±0.42)和(7.52±0.43)ng/μl。对照组和ALL组,ALL组化疗前和后,大便标本中乳酸杆菌和大肠埃希菌数量的对数值差异均有非常显著性(P<0.01)。结论HDMTX化疗ALL患儿肠道中乳酸杆菌和大肠埃希菌的数量较正常对照组明显减少。在HDMTX化疗治疗过程中,采用微生态制剂,调整细菌比例,使肠道内益生菌的数量尽快恢复正常是必要的。16S rRNA/DNA荧光定量PCR技术在临床细菌定量分析中切实可行。Objectives To study the variation of intestinal lactobacillus in children with acute lymphoblastic leukemia（ALL） and compare with the healthy children. To analyze the relationship between HDMTX and changes of intestinal microflora from microecology. To provide basis for preventing and treating ALL with microecologic agents. To investigate the practical fluorescent quantitative 16S rRNA/DNA-targeted PCR in quantitative study on bacterium. Methods The faces of 36 children with ALL of pre- and post-HDMTX therapy and 36 control children were collected. All the bacteria DNA in stool was extracted. The primes of Lactobacillua and Eseherichia coli with the 16S rRNA/DNA sequence of bacteria was designed. The quality of the bacteria through general PCR was obtained. The standard curve of fluorescent using a series of accurate bacteria DNA was produced. The amount of stool Lactobacillus and Escherichia coli among 36 control group and 36 children with ALL of pre- and post-HDMTX therapy were obtained by compared with the standard curve. Results Stool bacterial A260 of first day before treatment, of third day after treatment, of seventh day after treatment in patients with ALL of HDMTX and the control were （ 2436.3±768.6）, （ 1496.5±577. 1 ）, （ 1966.6 ±598.3 ）, （ 3479.3 ± 870.5 ） ng/μl respectively. Compared with the control children, the amount of Lactobacillus and Escherichia coli had both decrease in patients with ALL （P 〈 0.01 ）. Lactobacillus logarithmic absolute value of first day before treatment,of third day after treatment, of seventh day after treatment in patients with ALL and the control were （8.10 ±0.43） ,（ 6.73±0.45） ,（7.45±0.43） ,（9.12±0.50） ng/μl respectively （ P 〈 0.01 ）, Escherichia coli were （ 6.62 ± 0.4 ）, （ 5.96 ± 0.42 ）, （ 7.02 ± 0.42 ）, （ 7.52 ± 0.43 ） ng/μl respectively （ P 〈 0.01 ）. Conclusions The amount of Lactobacillus and Escherichia coli in patients with ALL of HD- MTX was less than the control. The microecologie

关 键 词：荧光定量 PCR 甲氨喋呤 急性淋巴细胞白血病 乳酸杆菌 大肠埃希菌 

分 类 号：R733.71[医药卫生—肿瘤]