GR基因多态性及其转录子与儿童急性淋巴细胞白血病GC抵抗的相关性研究

发布时间：2018-05-29 23:01

本文选题：儿童急性淋巴细胞白血病 + 基因多态性　；参考：《吉林大学》2015年博士论文

【摘要】：研究背景:糖皮质激素(GC)耐药是临床急性淋巴细胞白血病(ALL)治疗中常见的难题,也是导致治疗失败的主要原因之一。GC耐药机制复杂,目前为止尚未明确,GR作为GC的直接作用分子成为GC耐药的研究热点。GR基因编码区的单核苷酸多态性(SNP)位点在多种疾病中均与GC敏感性相关,同时体外实验研究表明在白血病细胞株中GR基因5'及3'端的转录子差异表达与GC敏感性相关。但针对GR基因非编码区SNP位点与儿童ALL糖皮质激素敏感性研究较少,另外在儿童ALL原代细胞中GR基因5'及3'端的转录子差异表达是否与GC敏感性相关。本研究试图探讨上述问题,本课题的完成将进一步揭示GC抵抗的分子机制,可能为GC的个体化治疗提供科学依据。研究一:GR基因多态性与儿童急性淋巴细胞白血病体内糖皮质激素反应的相关性研究目的:探讨GR基因单核苷酸多态性(包括编码区和非编码区)及其构成的单倍型与儿童急性淋巴细胞白血病体内糖皮质激素反应的相关性。方法:1.以63例儿童ALL患者和33例同龄健康儿童为研究对象。ALL患儿依据治疗最初一周强的松窗口试验结果分为反应良好组(PGR)及反应不良组(PPR)。采用病例对照研究的方法,研究GR基因多态性与中国汉族人群儿童ALL患者体内GC反应的相关性。本研究共选择5个SNP位点(rs7701443、rs10052957、rs41423247、rs6189/6190和rs6198)。2.争得家属及患儿本人同意,签署知情同意书,采集标本,初诊ALL儿童采集骨髓1ml,健康体检儿童标本收集体检剩余的血样(外周血200ul),进行基因组DNA提取,运用普通PCR及基因测序技术检测SNP位点的基因型。3.应用SPSS16.0软件进行统计分析,应用拟合优度χ2检验SNP位点基因型分布频率是否符合哈迪-温伯格遗传平衡定律,采用χ2检验法比较两组间等位基因频率、基因型频率的分布差异,并用Logistic回归做相关分析,应用SHEsis软件进行单倍型分析,检验水准α=0.05。结果:1.所研究的5个SNP位点在本研究人群中出现的频率GR基因非编码区的3个SNP位点(rs7701443,rs10052957,rs41423247)在本研究人群中均存在多态性,三者在病例组MAF分别为0.404、0.103、0.246,对照组MAF分别为0.454、0.09、0.287,基因型分布频率符合哈-温平衡。其余两个位点在病例组及对照组中均未检测到多态性。2.多态性位点rs41423247、rs7701443及rs10052957与儿童ALL体内强的松反应的相关性位点rs41423247在PGR及PPR两组间等位基因频率的分布差异具统计学意义,odds ratio(OR)=9.58,95%可信区间(CI):1.23-74.21,P=0.01;两组间基因型频率的分布无明显差异(P0.05);合并CG+GG基因型,与CC基因型相比,两组间分布差异具统计学意义,OR=9.778,95%CI:1.174-81.433,P=0.032。位点rs7701443在PGR及PPR两组间等位基因频率的分布差异具统计学意义,OR=3.12,95%CI:1.08-9,P=0.029;两组基因型频率分布差异无统计学意义。位点rs10052957等位基因及基因型频率在两组间的分布无明显差异(P0.05)。3.单倍型(rs7701443、rs10052957、rs41423247三个位点构成)与儿童ALL体内强的松反应的相关性由上述3个SNP位点构成的频率大于3%的单倍型共有4个,分别为CCC、TCG、TCC和TTG。单倍型CCC在PPR组的分布频率明显高于PGR组(P=0.013);而单倍型TCG在PGR组的分布频率明显高于PPR组(P=0.028)。结论:GR基因非编码区单核苷酸多态性位点(rs7701443、rs41423247)及其所构建的单倍型可能与中国东北地区汉族人群儿童急性淋巴细胞白血病体内强的松反应相关。研究二:GR基因转录子与儿童ALL原代细胞体外GC抵抗的相关性研究目的:探讨GR基因5'及3'端转录子的差异表达与儿童ALL原代细胞体外对GC抵抗的相关性。方法:1.以初次确诊为急性淋巴细胞白血病患者为研究对象,在争得家属及患儿本人同意及签署知情同意书的前提下,采集骨髓标本1ml用于后续研究。2.儿童ALL原代细胞进行体外培养(96孔板),体外给予prednisolone进行细胞毒试验,应用CCK-8法检测细胞增殖情况并计算IC50值,依据IC50值分为GC敏感组和耐药组(每组9个样本)。3.另一部分原代白血病细胞进行体外培养(24孔板),同样给予prednisolone干预,分别在干预的H0、H3、H8、H24四个时间点收集细胞,提取总RNA,逆转录成c DNA,然后应用实时定量PCR的方法对GR基因5'及3'端转录子(1A、1B、1C、GRα、GRβ)进行相对定量检测。4.应用SPSS16.0软件进行统计分析,采用均值±标准差形式表示数据,两组均数比较采用t检验,多组均数比较用方差分析,组间比较采用S-N-K法。结果:1.GC体外诱导ALL原代细胞GR-1A、1B、1C转录子表达变化与GC抵抗的相关性。体外给予prednisolone作用后,GC敏感组和GC耐药组外显子1A、1B、1C均表达上调,H24时间点上调水平最明显(P0.001)。以H24时间点表达量为研究对象,GC敏感组与耐药组相比外显子1A、1B、1C的表达上调幅度无明显差异(P0.05)。且GC敏感组及耐药组1A、1B、1C的差异表达无统计学意义(P0.05)。2.GC体外诱导儿童ALL原代细胞GRα、GRβ转录子的表达变化与GC抵抗的相关性。体外给予prednisolone作用后,GC敏感组和GC耐药组GRα、GRβm RNA表达均上调,H24时间点上调水平最明显(P0.001)。以H24时间点表达量为研究对象,GC敏感组GRαm RNA表达上调幅度明显高于GC耐药组(P=0.026),GRβm RNA的表达变化在两组间无明显差异(P0.05)。且GC敏感组及耐药组GRα的表达明显高于GRβ(P0.05)。结论:GC在体外可诱导儿童ALL原代细胞GR基因转录子1A、1B、1C、GRα、GRβm RNA表达上调,GC诱导GRα的上调幅度可能与GC敏感性相关。
[Abstract]:Background: the resistance of Glucocorticoid (GC) is a common problem in the treatment of clinical acute lymphoblastic leukemia (ALL), and it is also one of the main causes of the failure of treatment..GC is a complex mechanism. So far, GR as a direct molecule of GC has become a hotspot in the research hotspot of GC tolerance, the single nucleotide polymorphisms of the.GR gene coding region. SNP) loci are associated with GC sensitivity in a variety of diseases. At the same time, in vitro studies have shown that the differential expression of the 5'and 3' transcripts of the GR gene in the leukemia cell line is related to the sensitivity of the GC sensitivity. However, the study on the SNP site of the non coding region of the GR gene and the susceptibility to the ALL glucocorticoid in children is less, and the GR gene 5'is in the children's ALL primary cells. This study will further reveal the molecular mechanism of GC resistance and may provide a scientific basis for the individualized treatment of GC. Study 1: phase 1: the phase of GR gene polymorphism and glucocorticoid response in children with acute lymphoblastic leukemia. Objective: To investigate the correlation between single nucleotide polymorphisms of GR gene (including coding and non coding regions) and the correlation between haplotype and glucocorticoid response in children with acute lymphoblastic leukemia. Methods: 1. children with 63 children and 33 healthy children of the same age were selected for the first week of treatment based on the first week of treatment for children with.ALL. The results of the pine window test were divided into good reaction group (PGR) and poor reaction group (PPR). A case-control study was used to study the correlation between GR gene polymorphism and GC response in children with ALL in Chinese Han population. A total of 5 SNP loci (rs7701443, rs10052957, rs41423247, rs6189/6190 and rs6198) were selected for families and patients. The child himself agreed to sign the informed consent book, collect the specimen, collect the bone marrow 1ml for the first ALL children, collect the remaining blood samples of the physical examination (200ul) for the physical examination, extract the genomic DNA, and use the SPSS16.0 software of the common PCR and gene sequencing technology to detect the genotype.3. of the SNP site, and apply the goodness of fit. Whether the genotype distribution frequency of SNP locus was consistent with Hardy Weinberg's law of genetic balance, x 2 test was used to compare the allele frequency of two groups and the distribution difference of genotype frequency, and the correlation analysis was made by Logistic regression. The haplotype analysis was carried out by SHEsis software to test the level of alpha =0.05. results: 1. of the 5 SNP sites studied. 3 SNP loci (rs7701443, rs10052957, rs41423247) in the non coding region of the frequency GR gene (rs7701443, rs10052957, rs41423247) in this study population were polymorphic. The three in the case group were 0.404,0.103,0.246, the control group MAF was 0.454,0.09,0.287, and the genotype frequency was in accordance with the HA temperature balance. The other two loci were in the case. The polymorphic.2. polymorphism site rs41423247 was not detected in both group and control group, and the distribution of allele frequencies of rs7701443 and rs10052957 with ALL in ALL in children was statistically significant in the distribution of allele frequencies between groups of PGR and PPR two, odds ratio (OR) = 9.58,95% confidence interval; two groups There was no significant difference in the distribution of the type frequency (P0.05). The distribution of the two groups was statistically significant in the combination of the CG+GG genotype and the CC genotype. The distribution difference between the OR=9.778,95%CI:1.174-81.433 and P=0.032. loci rs7701443 in the PGR and PPR two groups was statistically significant, OR=3.12,95%CI:1.08-9, P=0.029, and two groups of genotype frequency. There was no significant difference in rate distribution. There was no significant difference in the distribution of allele rs10052957 alleles and genotype frequencies between the two groups (P0.05).3. haplotype (rs7701443, rs10052957, rs41423247 three loci) and the correlation of the reaction between the 3 SNP loci of the children and the haplotype of the 3 SNP loci above 3% of the above 3 SNP loci. The frequency of CCC, TCG, TCC and TTG. haplotype CCC in PPR group was significantly higher than that of PGR group (P=0.013), but the frequency of haplotype TCG in PGR group was significantly higher than that of PPR group (P=0.028). The correlation of prednisone response in the group of children with acute lymphoblastic leukemia. Study two: a study of the correlation between GR gene transcription and children's ALL primary cells in vitro GC resistance. Objective: To investigate the differential expression of GR gene 5'and 3' terminal transcripts and the correlation of GC resistance in children with ALL primary cells in vitro. Methods: 1. for the initial diagnosis of acute lymphatic lymph nodes On the premise of obtaining the consent and signing informed consent of the family and children, 1ml was used for the follow-up study of.2. children's ALL primary cells in vitro (96 orifice plates), and the cytotoxic test was given to prednisolone in vitro. The proliferation of cells was detected by CCK-8 and the IC50 value was calculated. According to the IC50 value, the GC sensitive group and the drug resistant group (each group of 9 samples).3. another part of the primary leukemia cells were cultured in vitro (24 orifice plates), and the prednisolone intervention was also given. The cells were collected at the four time points of H0, H3, H8, H24, respectively. The total RNA was extracted and the reverse transcript was transcribed to C DNA. "1A, 1B, 1C, GR a, GR beta) for relative quantitative detection of.4. application SPSS16.0 software for statistical analysis, using mean mean standard deviation to express the data. The average number of two groups was compared with t test, the multiple groups were compared with the variance analysis, and the group was compared with the S-N-K method. The result: 1.GC in vitro induced ALL primary cell GR-1A, transcription and transcription. The correlation between changes and GC resistance. After the effect of prednisolone in vitro, the expression of 1A, 1B, 1C in the GC sensitive group and the GC resistant group was up, and the time point up regulation of H24 was the most obvious (P0.001). The H24 time point expression was the research object, the GC sensitive group was compared with the drug resistance group. The differential expression of 1A, 1B and 1C in the sensitive and drug resistant groups was not statistically significant (P0.05).2.GC in vitro induced GR alpha in children ALL primary cells and the correlation between the expression of GR beta transcript and GC resistance. After the effect of prednisolone, GC sensitive and GC resistance groups were up up and the most obvious up level was up. The expression of GR alpha m RNA expression in the GC sensitive group was significantly higher than that in the GC group (P=0.026), and the expression of GR beta m RNA was not significantly different between the two groups (P0.05). The expression of 1C, GR alpha, GR beta m RNA was upregulated, and GC induced GR GR up regulation might be related to GC sensitivity.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R733.71

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