原发性膀胱输尿管反流Pax2基因启动子DNA甲基化状态研究

发布时间：2018-06-20 04:28

本文选题：膀胱输尿管反流 + Pax2　；参考：《复旦大学》2012年硕士论文

【摘要】：研究背景原发性膀胱输尿管反流(VUR)是一种常见的先天性尿路畸形,严重者可进展为终末期肾病(ESRD)。原发性VUR有一定的遗传基础,在同胞中的发病率显著高于健康儿童。国内外学者通过动物模型研究发现了一系列参与泌尿系统发育的基因与VUR发病相关,但对原发性VUR患者进行基因检测或全基因组扫描却未发现一致的基因异常,原发性VUR确切的发病机制迄今仍不明确。 Pax2基因所编码的蛋白质为核转录因子。在肾、输尿管发育过程中,Pax2表达于发育中的前肾、中肾、输尿管芽及间充质细胞,主要发挥三大作用,包括协调输尿管芽的出芽和定位,抑制输尿管芽的凋亡而促使输尿管芽不断发出分支,以及促使间充质细胞向上皮细胞转化。当上皮前体细胞形成后,Pax2表达逐渐下降,在成熟的肾单位无表达或呈痕量表达。我科前期通过免疫组织化学研究发现原发性VUR患儿输尿管上皮细胞Pax2蛋白阳性表达,而对照组呈痕量表达,且有研究报道在小鼠胚胎肾发育过程中Pax2表达逐渐下降的同时伴有其启动子某些特定区域DNA甲基化水平的增高。我们推测,Pax2在VUR患儿输尿管上皮细胞的表达可能与其启动子DNA甲基化的调控相关。在上述认识的基础上,本研究通过对Pax2启动子DNA甲基化水平的分析,探讨DNA甲基化对Pax2基因表达的影响及其表达的意义,为VUR发病机制的研究提供新的思路。目的(1)检测Pax2基因在人输尿管组织的表达情况。(2)检测Pax2基因启动子DNA甲基化水平。(3)通过检测DNA甲基转移酶(Dnmt)的表达分析引起DNA甲基化差异的可能原因。材料和方法(1)材料：病例组标本21例,来自我院泌尿外科行输尿管再植术的Ⅲ级以上原发性VUR患儿术中切下的异常输尿管下段组织,对照组3例,分别为外伤行肾、输尿管切除术,左输尿管结石伴末端狭窄行输尿管再植术,及肾移植术中取得的输尿管下段组织。(2)方法：通过免疫组织化学、Western blot及Real-time PCR检测VUR及对照组输尿管组织蛋白质及mRNA的表达,然后通过重亚硫酸盐测序(BSP)方法检测VUR及对照组输尿管组织Pax2基因启动子DNA甲基化水平,最后通过Real-time PCR检测VUR及对照组输尿管组织三种DNA甲基转移酶的mRNA表达。结果(1)Pax2表达于VUR输尿管上皮细胞胞核,且VUR组Pax2蛋白质(P=0.011)及mRNA(P0.0001)表达水平显著高于对照组。(2)Pax2的蛋白质表达量在不同年龄(P=0.309)、性别(P=0.316)及反流等级(P=0.339)的VUR中无显著差异。(3)VUR组Pax2基因所测启动子序列的DNA甲基化水平显著低于对照组(P=0.045),但与mRNA的表达无显著相关性(P=0.091)。(4)Pax2基因所测启动子序列的DNA甲基化水平在不同年龄(P=0.574)、性别(P=0.347)、反流等级(P=0.881)的VUR中无显著差异。(5)VUR组Dnmt3a显著低于对照组(P=0.027),但Dnmtl、Dnmt3b的差异无统计学意义。结论(1)VUR组Pax2基因蛋白质及mRNA的表达显著高于对照组,其表达的意义有待进一步研究。(2)VUR组Pax2基因所测启动子序列的DNA甲基化水平显著低于对照组,在一定程度上可以解释Pax2在输尿管上皮细胞的高表达。(3)Pax2基因所测启动子的DNA低甲基化可能是由低水平的Dnmt3a所引起。
[Abstract]:Research background
Primary cysturreteral reflux (VUR) is a common congenital urinary tract malformation, and the serious person can advance to end-stage renal disease (ESRD). Primary VUR has a certain genetic basis, and the incidence of the disease is significantly higher in the siblings than in healthy children. The incidence of R is related, but genetic abnormalities are not found in primary VUR patients or all genome scans, and the exact pathogenesis of primary VUR is still unclear.
The protein encoded by the Pax2 gene is a nuclear transcription factor. During the development of the kidney and ureter, Pax2 is expressed in the developing anterior kidney, middle kidney, ureter buds and mesenchymal cells, which mainly play three major roles, including coordinating the buds and locating of ureter buds, inhibiting the apoptosis of ureteral buds and promoting the continuous branching of ureteral buds, and promoting the development of ureteral buds. Mesenchymal cells were transformed into epithelial cells. After the formation of epithelial progenitor cells, the expression of Pax2 gradually decreased, and no expression or trace expression was observed in mature nephron.
The positive expression of Pax2 protein in the ureteral epithelial cells of the children with primary VUR was found in the early stage of the immuno histochemical study, while the control group showed a trace expression, and the study reported that the expression of Pax2 decreased gradually during the development of mouse embryonic kidney accompanied by the increase of the level of DNA methylation in certain specific regions of the promoter. We speculated that Pa The expression of x2 in the ureteral epithelial cells in children with VUR may be related to the regulation of the promoter DNA methylation. On the basis of the above understanding, this study explores the effect of DNA methylation on the expression of Pax2 gene and its significance by analyzing the DNA methylation level of the Pax2 promoter, providing a new idea for the study of the pathogenesis of VUR.
Objective (1) to detect the expression of Pax2 gene in human ureteral tissue. (2) to detect the level of DNA methylation of the Pax2 gene promoter. (3) the possible reasons for the difference of DNA methylation by detecting the expression of DNA methyltransferase (Dnmt).
Materials and methods (1) material: 21 cases of case group specimens from our hospital department of Urology for ureteral reimplantation of primary VUR children with abnormal ureteral lower segment, 3 cases of the control group, ureterectomy, ureterectomy, left ureteral stony with terminal stricture, and renal transplantation. The lower ureter tissues were obtained. (2) the expression of protein and mRNA in the ureteral tissues was detected by immunohistochemistry, Western blot and Real-time PCR, and then the Pax2 gene promoter DNA methylation level of the ureter tissue in VUR and the control group was detected by the heavy sulfite sequencing (BSP) method, and the Real-time P was finally passed through Real-time P. CR was used to detect the mRNA expression of three DNA methyltransferases in VUR and control group.
Results (1) Pax2 was expressed in the nucleus of VUR ureteral epithelial cells, and the expression level of Pax2 protein (P=0.011) and mRNA (P0.0001) in VUR group was significantly higher than that of the control group. (2) there was no significant difference in the protein expression of Pax2 at different ages (P=0.309), sex (P=0.316) and reflux grade (P=0.339) VUR. The level of methylation was significantly lower than that of the control group (P=0.045), but there was no significant correlation with the expression of mRNA (P=0.091). (4) there was no significant difference in the DNA methylation level of the promoter sequence of the Pax2 gene at different ages (P=0.574), sex (P=0.347), and the regurgitation grade (P=0.881). (5) VUR Dnmt3a was significantly lower than the control group (P=0.027). The difference was not statistically significant.
Conclusion (1) the expression of Pax2 gene protein and mRNA in VUR group is significantly higher than that of the control group, and the significance of the expression needs further study. (2) the DNA methylation level of the promoter sequence of the Pax2 gene in the VUR group is significantly lower than that of the control group. To a certain extent, the high expression of Pax2 in the ureteral epithelial cells is explained. (3) DN of the promoter measured by the Pax2 gene. A hypomethylation may be caused by low level Dnmt3a.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R726.9

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