内皮素1基因多态性与儿童青少年骨肉瘤化疗耐药的相关性分析

发布时间：2018-05-22 20:43

  本文选题：内皮素1 + 基因多态性　； 参考：《中南大学》2014年博士论文

【摘要】：目的：本研究首次在病例对照研究中探索ET-1的SNP位点和单倍型与儿童及青少年骨肉瘤的化疗耐药风险之间的关系。为证明ET-1基因的SNP和单倍型与儿童及青少年骨肉瘤化疗耐药的风险变化之间存在相关关系,提供了第一手的证据,从而为化疗耐药的儿童及青少年骨肉瘤患者在病理生理学和治疗上提出了全新的观念。 方法：本研究分三部分；1.用350对年龄、性别、肿瘤位置和发展阶段相匹配的儿童青少年骨肉瘤患者进行研究。2.苯酚/氯仿法从白细胞中分离出基因组DNA,并存储在400毫升的TE中(10mMTris/HCl和1mM EDTA的溶液(pH8.0)。 ET-1基因单核苷酸多态性的检测是通过ABI3700DNA分析仪(Applied Biosystems)上进行毛细管电泳寡核苷酸连接测定。对检测结果进行Hardy-Weinberg平衡检验、将rs1800541和rs2070699进行配对LD试验,计算使用HaploView程序,并对所得进行统计学分析；3.将患者手术切除的骨肉瘤标本进行POCC,对POCC进行传代培养,实时定量逆转录聚合酶链反应(RT-PCR)测定ET-1mRNA水平,并通过酶联免疫吸附试验(ELISA)对POCC上清液中细胞分泌的ET-1水平进行测定,观察肿瘤坏死百分比。 结果： 1.发现在年龄、性别及肿瘤位置和发展阶段没有显著的差异(表1)。同时在病例组和对照组之间,在体重指数(BMI)、收缩压、舒张压、血浆ET-1的水平亦无显著差异(表1)。 2.在三个SNPs中,对照组中的rs5370明显偏离了Hardy-Weinberg平衡。 3.在rs1800541上的G等位基因与新辅助化疗的弱反应或者化学耐药性的风险降低有显著相关性(P0.01),而位于rs2070699上的G等位基因与耐药风险增加相关的倾向并不明显,P值位于临界水平(P=0.06)。 4.rs1800541和rs2070699存在较强LD关系(病例组,D'=0.940,r2=0.885；对照组,D'=0.925,r2=0.860)。 5.2-SNP单倍型TG在病例组中比对照组的显著相关性更常见(P=0.012,校正OR,1.82,95％CI,1.10-5.65),而2-SNP单倍型GT在对照组中比病例组中的显著相关性更常见(P=0.009,调整后的 OR,0.25,95％CI,0.14-0.84)。 6.在化疗耐药儿童及青少年骨肉瘤患者中,骨肉瘤细胞分泌的ET-1 水平与肿瘤坏死的百分比具有显著的负相关关系。 结论： 1.儿童青少年骨肉瘤患者在化疗后肿瘤坏死与年龄,性别及肿瘤的位置及阶段、体重指数(BMI)、收缩压、舒张压、血浆ET-1无相关性。 2.rs5370与儿童青少年骨肉瘤化疗耐药的风险无明显相关性。 3.rs1800541上的G等位基因与新辅助化疗的弱反应或者化学耐药性的风险降低有显著相关性,而位于rs2070699上的G等位基因与耐药性风险增加有相关性的倾向不明显。 4.rs1800541和rs2070699存在强LD关系。 5.TG单倍型和GT单倍型分别与耐药性儿科骨肉瘤的增高风险和降低风险有关系。 6.在儿童及青少年骨肉瘤化疗耐药患者中,骨肉瘤细胞分泌的ET-1水平与肿瘤坏死的百分比具有显著的负相关关系。
[Abstract]:Objective: To explore the relationship between the SNP locus and haplotype of ET-1 and the risk of chemotherapeutic resistance in children and adolescents with osteosarcoma for the first time in a case-control study. The first hand evidence is provided to demonstrate the correlation between the risk of SNP and haplotype of ET-1 gene and the risk of chemotherapeutic resistance to osteosarcoma in children and adolescents. For children and adolescents with chemotherapy resistant osteosarcoma, a new concept has been put forward in pathophysiology and treatment.
Methods: This study was divided into three parts. 1. the genomic DNA of children with osteosarcoma matched in age, sex, tumor location and development phase was studied by.2. phenol / chloroform method from white cells, and stored in 400 ml of TE (10mMTris/HCl and 1mM EDTA solution (pH8.0). ET-1 gene single nucleotide polymorphisms. The detection was carried out by the ABI3700DNA analyzer (Applied Biosystems) on the capillary electrophoresis oligonucleotide connection. The results were tested by Hardy-Weinberg balance test, rs1800541 and rs2070699 were paired LD test, HaploView program was used, and the results were statistically analyzed. 3. the osteosarcoma resection of the patients was performed. The specimens were POCC, POCC was cultured, real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine the level of ET-1mRNA, and the level of ET-1 secreted in POCC supernatant was measured by enzyme linked immunosorbent assay (ELISA), and the percentage of tumor necrosis was observed.
Result锛，

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