本文選題：兒童急性淋巴細胞白血病 + 基因多態(tài)性�。� 參考：《吉林大學(xué)》2015年博士論文

【摘要】：研究背景:糖皮質(zhì)激素(GC)耐藥是臨床急性淋巴細胞白血病(ALL)治療中常見的難題,也是導(dǎo)致治療失敗的主要原因之一。GC耐藥機制復(fù)雜,目前為止尚未明確,GR作為GC的直接作用分子成為GC耐藥的研究熱點。GR基因編碼區(qū)的單核苷酸多態(tài)性(SNP)位點在多種疾病中均與GC敏感性相關(guān),同時體外實驗研究表明在白血病細胞株中GR基因5'及3'端的轉(zhuǎn)錄子差異表達與GC敏感性相關(guān)。但針對GR基因非編碼區(qū)SNP位點與兒童ALL糖皮質(zhì)激素敏感性研究較少,另外在兒童ALL原代細胞中GR基因5'及3'端的轉(zhuǎn)錄子差異表達是否與GC敏感性相關(guān)。本研究試圖探討上述問題,本課題的完成將進一步揭示GC抵抗的分子機制,可能為GC的個體化治療提供科學(xué)依據(jù)。研究一:GR基因多態(tài)性與兒童急性淋巴細胞白血病體內(nèi)糖皮質(zhì)激素反應(yīng)的相關(guān)性研究目的:探討GR基因單核苷酸多態(tài)性(包括編碼區(qū)和非編碼區(qū))及其構(gòu)成的單倍型與兒童急性淋巴細胞白血病體內(nèi)糖皮質(zhì)激素反應(yīng)的相關(guān)性。方法:1.以63例兒童ALL患者和33例同齡健康兒童為研究對象。ALL患兒依據(jù)治療最初一周強的松窗口試驗結(jié)果分為反應(yīng)良好組(PGR)及反應(yīng)不良組(PPR)。采用病例對照研究的方法,研究GR基因多態(tài)性與中國漢族人群兒童ALL患者體內(nèi)GC反應(yīng)的相關(guān)性。本研究共選擇5個SNP位點(rs7701443、rs10052957、rs41423247、rs6189/6190和rs6198)。2.爭得家屬及患兒本人同意,簽署知情同意書,采集標(biāo)本,初診ALL兒童采集骨髓1ml,健康體檢兒童標(biāo)本收集體檢剩余的血樣(外周血200ul),進行基因組DNA提取,運用普通PCR及基因測序技術(shù)檢測SNP位點的基因型。3.應(yīng)用SPSS16.0軟件進行統(tǒng)計分析,應(yīng)用擬合優(yōu)度χ2檢驗SNP位點基因型分布頻率是否符合哈迪-溫伯格遺傳平衡定律,采用χ2檢驗法比較兩組間等位基因頻率、基因型頻率的分布差異,并用Logistic回歸做相關(guān)分析,應(yīng)用SHEsis軟件進行單倍型分析,檢驗水準(zhǔn)α=0.05。結(jié)果:1.所研究的5個SNP位點在本研究人群中出現(xiàn)的頻率GR基因非編碼區(qū)的3個SNP位點(rs7701443,rs10052957,rs41423247)在本研究人群中均存在多態(tài)性,三者在病例組MAF分別為0.404、0.103、0.246,對照組MAF分別為0.454、0.09、0.287,基因型分布頻率符合哈-溫平衡。其余兩個位點在病例組及對照組中均未檢測到多態(tài)性。2.多態(tài)性位點rs41423247、rs7701443及rs10052957與兒童ALL體內(nèi)強的松反應(yīng)的相關(guān)性位點rs41423247在PGR及PPR兩組間等位基因頻率的分布差異具統(tǒng)計學(xué)意義,odds ratio(OR)=9.58,95%可信區(qū)間(CI):1.23-74.21,P=0.01;兩組間基因型頻率的分布無明顯差異(P0.05);合并CG+GG基因型,與CC基因型相比,兩組間分布差異具統(tǒng)計學(xué)意義,OR=9.778,95%CI:1.174-81.433,P=0.032。位點rs7701443在PGR及PPR兩組間等位基因頻率的分布差異具統(tǒng)計學(xué)意義,OR=3.12,95%CI:1.08-9,P=0.029;兩組基因型頻率分布差異無統(tǒng)計學(xué)意義。位點rs10052957等位基因及基因型頻率在兩組間的分布無明顯差異(P0.05)。3.單倍型(rs7701443、rs10052957、rs41423247三個位點構(gòu)成)與兒童ALL體內(nèi)強的松反應(yīng)的相關(guān)性由上述3個SNP位點構(gòu)成的頻率大于3%的單倍型共有4個,分別為CCC、TCG、TCC和TTG。單倍型CCC在PPR組的分布頻率明顯高于PGR組(P=0.013);而單倍型TCG在PGR組的分布頻率明顯高于PPR組(P=0.028)。結(jié)論:GR基因非編碼區(qū)單核苷酸多態(tài)性位點(rs7701443、rs41423247)及其所構(gòu)建的單倍型可能與中國東北地區(qū)漢族人群兒童急性淋巴細胞白血病體內(nèi)強的松反應(yīng)相關(guān)。研究二:GR基因轉(zhuǎn)錄子與兒童ALL原代細胞體外GC抵抗的相關(guān)性研究目的:探討GR基因5'及3'端轉(zhuǎn)錄子的差異表達與兒童ALL原代細胞體外對GC抵抗的相關(guān)性。方法:1.以初次確診為急性淋巴細胞白血病患者為研究對象,在爭得家屬及患兒本人同意及簽署知情同意書的前提下,采集骨髓標(biāo)本1ml用于后續(xù)研究。2.兒童ALL原代細胞進行體外培養(yǎng)(96孔板),體外給予prednisolone進行細胞毒試驗,應(yīng)用CCK-8法檢測細胞增殖情況并計算IC50值,依據(jù)IC50值分為GC敏感組和耐藥組(每組9個樣本)。3.另一部分原代白血病細胞進行體外培養(yǎng)(24孔板),同樣給予prednisolone干預(yù),分別在干預(yù)的H0、H3、H8、H24四個時間點收集細胞,提取總RNA,逆轉(zhuǎn)錄成c DNA,然后應(yīng)用實時定量PCR的方法對GR基因5'及3'端轉(zhuǎn)錄子(1A、1B、1C、GRα、GRβ)進行相對定量檢測。4.應(yīng)用SPSS16.0軟件進行統(tǒng)計分析,采用均值±標(biāo)準(zhǔn)差形式表示數(shù)據(jù),兩組均數(shù)比較采用t檢驗,多組均數(shù)比較用方差分析,組間比較采用S-N-K法。結(jié)果:1.GC體外誘導(dǎo)ALL原代細胞GR-1A、1B、1C轉(zhuǎn)錄子表達變化與GC抵抗的相關(guān)性。體外給予prednisolone作用后,GC敏感組和GC耐藥組外顯子1A、1B、1C均表達上調(diào),H24時間點上調(diào)水平最明顯(P0.001)。以H24時間點表達量為研究對象,GC敏感組與耐藥組相比外顯子1A、1B、1C的表達上調(diào)幅度無明顯差異(P0.05)。且GC敏感組及耐藥組1A、1B、1C的差異表達無統(tǒng)計學(xué)意義(P0.05)。2.GC體外誘導(dǎo)兒童ALL原代細胞GRα、GRβ轉(zhuǎn)錄子的表達變化與GC抵抗的相關(guān)性。體外給予prednisolone作用后,GC敏感組和GC耐藥組GRα、GRβm RNA表達均上調(diào),H24時間點上調(diào)水平最明顯(P0.001)。以H24時間點表達量為研究對象,GC敏感組GRαm RNA表達上調(diào)幅度明顯高于GC耐藥組(P=0.026),GRβm RNA的表達變化在兩組間無明顯差異(P0.05)。且GC敏感組及耐藥組GRα的表達明顯高于GRβ(P0.05)。結(jié)論:GC在體外可誘導(dǎo)兒童ALL原代細胞GR基因轉(zhuǎn)錄子1A、1B、1C、GRα、GRβm RNA表達上調(diào),GC誘導(dǎo)GRα的上調(diào)幅度可能與GC敏感性相關(guān)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R733.71

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本文編號：1952792