大剂量甲氨蝶呤化疗对急性淋巴细胞白血病患儿肠道菌群的影响

目的 探讨大剂量甲氨蝶呤(HDMTX)化疗对急性淋巴细胞白血病(ALL)患儿肠道菌群的影响.方法 收集36名HDMTX化疗前后ALL患儿及36名健康对照组儿童的粪便标本,并提取目标细菌DNA.用细菌的16SrRNA/DNA序列设计双歧杆菌和大肠杆菌的引物,行常规PCR完成细菌的定性,然后取准确定量的2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2种细菌的量.结果 ALL组HDMTX化疗前1 d、化疗后第3、7天和对照组的肠道双歧杆菌数量的对数值分别为(7.24±0.33)拷贝数/克湿便、(6.00±0.27)拷贝数/克湿便、(6.59±0.33)拷贝数/克湿便和(9.49±0.41)拷贝数/克湿便;而大肠杆菌数量的对数值,ALL组化疗前1 d、化疗后第3、7天和对照组的分别是(6.62±0.42)拷贝数/克湿便,(5.96±0.42)拷贝数/克湿便,(7.02±0.41)拷贝数/克湿便,(7.52±0.43)拷贝数/克湿便.双歧杆菌和大肠杆菌对照组均较ALL组高(F=739.61,88.67,P均＜0.01).ALL组化疗前和后,大便标本中双歧杆菌和大肠杆菌数量变化差异有统计学意义(P均＜0.01),其中化疗第3天下降最明显,第7天有所回升.结论 (1)HDMTX化疗方案对ALL患儿肠道双歧杆菌和大肠杆菌有明显影响,化疗后数量显著下降;(2)ALL患儿肠道菌群和健康儿童相比,益生菌明显减少.

Effects of high-dose methotrexate therapy on intestinal bacterial flora in children with acute lymphoblastic leukemia

Objective To investigate the effects of high-dose methotrexate(HDMTX) therapy on intestinal bacterial flora in children with acute lymphoblastic leukemia(ALL).Methods Thirty-six children with ALL of pre-and post-HDMTX therapy and 36 control children were enrolled.The bacterial DNA in stool was extracted.The primers for Bacillus bifidus and Escherichia coli with the 16SrRNA/DNA sequence of bacteria were designed.The bacteria were identified through general PCR.The standard eurve of both bacterial DNA was produced using a series of dilution of accurately quantified bacteriaI DNA.The unknown samples were measured by 16SrRNA/DNA-targeted PCR.The amount of stool Bacillus bifidus and Escherichia coli among 36 control children and 36 children with ALL of pre-and post-HDMTX therapy were obtained by using the standard curves.Reslllts Bacillus bifidus logarithmic absolute value of the first day before treatment,of third day after treatment,of seventh day after treatment in patients with ALL and the control was 7.24±0.33,6.00±0.27,6.59±0.33,and 9.49±0.41,respectivelv(P<0.01).Escherichia coli logarithmic absolute value of the first day before treatment,of third day after treatment,of seventh day after treatment in patients with ALL and the control was 6.62±0.42,5.96±0.42,7.02±0.41,and 7.52±0.43,respectively(P<0.01).The logarithm of the amount of stool Bacillus bifidus and Escherichia coli in control group was higher in ALL group(F=739.61,88.67,P<0.01).There were significant differenee(P<0.01)in the logarithm of the amount of stool Bacillus bifidus and Escherichia coli between pre-therapy and post-therapy group.The logarithm of the bacterium was very low on third day after treatment,but gradually increased on the seventh day after treatment.Conclusions (1)HDMTX therapy has great effects on intestinal flora of Bacillus bifidus and Escherichia coli and significantly reduced the bacteria in children with ALL.(2)Probiotics had significantly decreased in ALL group than in control group.