【作者】 冯毅；

【导师】 于洁；

【作者基本信息】 重庆医科大学 ， 儿科学， 2010， 硕士

【摘要】 目的:①探讨汉族人群中多药耐药基因1(multidrug resistance gene 1,MDR1) C3435T位点多态性对外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)中mRNA表达的影响。②通过研究苯巴比妥(phenobarbitone,PB)对PBMCs MDR1表达的影响,了解PBMCs MDR1的表达是否也能被诱导,并研究了PBMCs中MDR1与孕烷X受体(pregnane X receptor,PXR)表达的关系,初步探讨其意义。方法:①以163名(男性98名,女性65名)无亲缘关系的中国健康汉族儿童的外周血标本为研究对象,采用PCR-RFLP技术检测其MDR1基因C3435T位点的基因型,实时荧光定量PCR法检测其PBMCs MDR1 mRNA表达水平。②将36名健康儿童的PBMCs分离后,将其分编为无培养组(不进行培养和药物处理)、自然培养组(只单纯培养不进行药物处理)及PB培养组(加入终浓度为40μm/L苯巴比妥处理)。将自然培养组与PB培养组培养24h后,实时荧光定量PCR检测这三组细胞的MDR1及PXR mRNA表达水平。③Hardy-Weinberg检验采用卡方检验。不同分组间的PBMCs MDR1或PXR表达水平的比较采用t检验或单因素方差分析,MDR1及PXR的表达水平相关性分析使用Spearman法。P<0.05表示差异有统计学意义。结果:①在163名健康中国汉族儿童中,63名为野生型纯合子3435CC型,78名为杂合子CT型,22名为突变型纯合子TT型,CC、CT与TT各基因型的发生频率分别为38.7%、47.9%与13.5%;其C等位基因频率为62.6%,T等位基因频率为37.4%;C3435T多态性位点的基因型频率分布符合Hardy-Weinberg平衡(P>0.05);CC、CT和TT各基因型组间PBMCs MDR1 mRNA表达水平分别为(4.562±3.383)×10-3、(4.673±3.710)×10-3和(4.489±2.928)×10-3,差异无统计学意义(P>0.05)。②未培养组、自发培养组和PB培养组MDR1 mRNA表达水平分别为(4.475±2.980)×10-3、(4.991±4.165)×10-3及(7.265±5.067)×10-3 , PXR mRNA在以上三组中的表达水平分别为(2.073±1.335)×10-3、(1.836±1.467)×10-3及(3.014±2.238)×10-3。PB培养组MDR1和PXR表达水平均高于未培养组和自发培养组,差异有统计学意义(P<0.05),而未培养组和自发培养组之间的MDR1和PXR表达水平差异无统计学意义(P>0.05);相关性分析提示,苯巴比妥处理后MDR1及PXR mRNA的表达水平呈正相关关系(r=0.517,P=0.0012)。结论:①汉族人群中MDR1基因C3435T多态性对PBMCs mRNA表达并没有显著性影响,对于汉族儿童C3435T位点多态性不能作为推测MDR1表达水平的依据。②苯巴比妥可诱导PBMCs MDR1表达,提示PBMCs MDR1表达也能被诱导。苯巴比妥诱导后PXR与MDR1的表达水平呈正相关关系,提示PXR可能参与了PBMCs MDR1基因的表达调控。更多还原

【Abstract】 Objective: To investigate the association of MDR1 C3435T polymorphism with its mRNA expression in peripheral blood mononuclear cells in healthy persons of Han populations. Furthermore, to investigate whether phenobarbital has effect on the expression of MDR1 in peripheral bloods mononuclear cells and try to demonstrate whether expression of MDR1 in PBMCs can be induced. After using phenobarbital the relationship between MDR1 and PXR mRNA expression levels in PBMCs was studied.Methods①163 (98males, 65females) blood samples were taken from unrelated Chinese Han healthy children. Genotypes of the MDR1 C3435T polymorphism were identified by PCR-RFLP. MDR1 mRNA expression levels in PBMCs were detected by SYBR green I real-time polymerase chain reaction.②The PBMCs were isolated and purified from 36 healthy children and then were divided into three groups for testing.The first group of PBMCs was not cultured and stimulated. The second group of PBMCs was cultured naturally.The third group of PBMC was stimulated by phenobarbital . After the PBMCs were cultured for 24 hours, the MDR1 and PXR mRNA expression levels were detected by SYBR green I real-time PCR.③Statistical methods: Hardy-Weinberg equilibrium testing was performed for the C3435T polymorphism using the chi-squared test. One way analysis of variance and t-test were used to compare the MDR1 or PXR mRNA expression levels among different groups.Spearman analysis was performed to investigate the correlation between MDR1 and PXR mRNA expression levels, accepting P<0.05 as being significant.Results①The CC, CT and TT genotype frequencies of C3435T polymorphism were 63(38.7%), 78(47.9%)and 22(13.5%), respectively. For C3435T polymorphism, the frequency of 3435C allele is 62.6%.The distribution of genotypes was consistent with Hardy-Weinberg equilibrium(P>0.05). The MDR1 mRNA expression levels of CC, CT and TT genotype groups were (4.562±3.383)×10-3, (4.673±3.710)×10-3and (4.489±2.928)×10-3, respectively.There were no significant differences were found among the three genotypes in PBMCs (P>0.05).②The MDR1 mRNA expression levels in uncultured PBMCs group, naturally cultured PBMCs group and the PBMCs group stimulated with PB were (4.475±2.980)×10-3,(4.991±4.165)×10-3and(7.265±5.067)×10-3,respectively.For PXR mRNA expression levels ,these were (2.073±1.335)×10-3, (1.836±1.467)×10-3 and (3.014±2.238)×10-3 in above groups,respectively. Compared with uncultured PBMCs group and naturally cultured PBMCs group,the MDR1 and PXR mRNA expression levels in the PBMCs group stimulated with PB were elevated significantly (P<0.05). There were no significant differences between the uncultured and naturally cultured PBMCs in MDR1 and PXR mRNA expression levels (P>0.05).The MDR1 mRNA expression levels were positively correlated with PXR expression after being stimulated by PB(r=0.517,P=0.0012).Conclusion①The results suggest that the MDR1 C3435T polymorphism does not appear to have a significant effect on its mRNA expression in PBMCs in Chinese Han populations. So that, the genotypes of C3435T polymorphism can not allow the predicting of the expression of MDR1 in Chinese Han children.②The phenobarbital can induce the expression of MDR1 in PBMCs ,which proved the expression of MDR1 in PBMCs can be induced. The MDR1 mRNA expression levels were positively correlated with PXR expression after being stimulated by Phenobarbital, which suggested that PXR may play some role in the regulation of MDR1 expression in PBMCs.更多还原