本文選題：膀胱輸尿管反流 + Pax2��； 參考：《復(fù)旦大學(xué)》2012年碩士論文

【摘要】：研究背景 原發(fā)性膀胱輸尿管反流(VUR)是一種常見的先天性尿路畸形,嚴(yán)重者可進(jìn)展為終末期腎病(ESRD)。原發(fā)性VUR有一定的遺傳基礎(chǔ),在同胞中的發(fā)病率顯著高于健康兒童。國內(nèi)外學(xué)者通過動物模型研究發(fā)現(xiàn)了一系列參與泌尿系統(tǒng)發(fā)育的基因與VUR發(fā)病相關(guān),但對原發(fā)性VUR患者進(jìn)行基因檢測或全基因組掃描卻未發(fā)現(xiàn)一致的基因異常,原發(fā)性VUR確切的發(fā)病機(jī)制迄今仍不明確。 Pax2基因所編碼的蛋白質(zhì)為核轉(zhuǎn)錄因子。在腎、輸尿管發(fā)育過程中,Pax2表達(dá)于發(fā)育中的前腎、中腎、輸尿管芽及間充質(zhì)細(xì)胞,主要發(fā)揮三大作用,包括協(xié)調(diào)輸尿管芽的出芽和定位,抑制輸尿管芽的凋亡而促使輸尿管芽不斷發(fā)出分支,以及促使間充質(zhì)細(xì)胞向上皮細(xì)胞轉(zhuǎn)化。當(dāng)上皮前體細(xì)胞形成后,Pax2表達(dá)逐漸下降,在成熟的腎單位無表達(dá)或呈痕量表達(dá)。 我科前期通過免疫組織化學(xué)研究發(fā)現(xiàn)原發(fā)性VUR患兒輸尿管上皮細(xì)胞Pax2蛋白陽性表達(dá),而對照組呈痕量表達(dá),且有研究報(bào)道在小鼠胚胎腎發(fā)育過程中Pax2表達(dá)逐漸下降的同時伴有其啟動子某些特定區(qū)域DNA甲基化水平的增高。我們推測,Pax2在VUR患兒輸尿管上皮細(xì)胞的表達(dá)可能與其啟動子DNA甲基化的調(diào)控相關(guān)。在上述認(rèn)識的基礎(chǔ)上,本研究通過對Pax2啟動子DNA甲基化水平的分析,探討DNA甲基化對Pax2基因表達(dá)的影響及其表達(dá)的意義,為VUR發(fā)病機(jī)制的研究提供新的思路。 目的(1)檢測Pax2基因在人輸尿管組織的表達(dá)情況。(2)檢測Pax2基因啟動子DNA甲基化水平。(3)通過檢測DNA甲基轉(zhuǎn)移酶(Dnmt)的表達(dá)分析引起DNA甲基化差異的可能原因。 材料和方法(1)材料：病例組標(biāo)本21例,來自我院泌尿外科行輸尿管再植術(shù)的Ⅲ級以上原發(fā)性VUR患兒術(shù)中切下的異常輸尿管下段組織,對照組3例,分別為外傷行腎、輸尿管切除術(shù),左輸尿管結(jié)石伴末端狹窄行輸尿管再植術(shù),及腎移植術(shù)中取得的輸尿管下段組織。(2)方法：通過免疫組織化學(xué)、Western blot及Real-time PCR檢測VUR及對照組輸尿管組織蛋白質(zhì)及mRNA的表達(dá),然后通過重亞硫酸鹽測序(BSP)方法檢測VUR及對照組輸尿管組織Pax2基因啟動子DNA甲基化水平,最后通過Real-time PCR檢測VUR及對照組輸尿管組織三種DNA甲基轉(zhuǎn)移酶的mRNA表達(dá)。 結(jié)果(1)Pax2表達(dá)于VUR輸尿管上皮細(xì)胞胞核,且VUR組Pax2蛋白質(zhì)(P=0.011)及mRNA(P0.0001)表達(dá)水平顯著高于對照組。(2)Pax2的蛋白質(zhì)表達(dá)量在不同年齡(P=0.309)、性別(P=0.316)及反流等級(P=0.339)的VUR中無顯著差異。(3)VUR組Pax2基因所測啟動子序列的DNA甲基化水平顯著低于對照組(P=0.045),但與mRNA的表達(dá)無顯著相關(guān)性(P=0.091)。(4)Pax2基因所測啟動子序列的DNA甲基化水平在不同年齡(P=0.574)、性別(P=0.347)、反流等級(P=0.881)的VUR中無顯著差異。(5)VUR組Dnmt3a顯著低于對照組(P=0.027),但Dnmtl、Dnmt3b的差異無統(tǒng)計(jì)學(xué)意義。 結(jié)論(1)VUR組Pax2基因蛋白質(zhì)及mRNA的表達(dá)顯著高于對照組,其表達(dá)的意義有待進(jìn)一步研究。(2)VUR組Pax2基因所測啟動子序列的DNA甲基化水平顯著低于對照組,在一定程度上可以解釋Pax2在輸尿管上皮細(xì)胞的高表達(dá)。(3)Pax2基因所測啟動子的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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R726.9

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