宮內(nèi)發(fā)育遲緩大鼠WT1和IGF2 DNA甲基化與腎臟發(fā)育和功能的關(guān)系研究
發(fā)布時(shí)間：2018-07-15 21:28
【摘要】：各種研究顯示宮內(nèi)營(yíng)養(yǎng)不良可以持續(xù)改變機(jī)體的結(jié)構(gòu)、功能和代謝,引起宮內(nèi)發(fā)育遲緩(intrauterine growth retardation, IUGR),而且可能通過“發(fā)育程序化”途徑,成為成年期發(fā)生高血壓、冠心病,胰島素抵抗、2型糖尿病、肥胖和慢性腎臟病等的重要危險(xiǎn)因素。腎臟是“發(fā)育程序化”的重要靶器官,流行病學(xué)資料顯示,IUGR胎兒腎臟體積較正常胎兒明顯縮小同時(shí)腎小球數(shù)目明顯減少；生后長(zhǎng)期隨訪發(fā)現(xiàn),IUGR組腎小球?yàn)V過功能較正常組明顯降低而蛋白尿和終末期腎臟疾病的發(fā)生率明顯增高。我們課題組前期動(dòng)物實(shí)驗(yàn)同樣發(fā)現(xiàn),IUGR新生鼠腎小球數(shù)目明顯減少,且生后隨訪中蛋白尿和高血壓的發(fā)生率明顯增高。但是“發(fā)育程序化”腎臟疾病的確切分子機(jī)制目前仍未闡明。 研究報(bào)道Wilms瘤1基因(WT1)和胰島素樣生長(zhǎng)因子2(IGF2)是影響腎臟發(fā)育的重要基因,對(duì)腎單位的發(fā)生和腎小球的濾過功能起到了重要作用。我們前期研究業(yè)已證實(shí),IUGR新生大鼠WT1基因表達(dá)明顯下調(diào)。因此,我們推測(cè)在“發(fā)育程序化”腎臟疾病的發(fā)生過程中,WT1和IGF2可能是引起生后腎小球數(shù)目減少等腎臟病變的關(guān)鍵分子。在發(fā)育過程中,由于表觀遺傳可調(diào)控基因啟動(dòng)子區(qū)并且維持其作用在機(jī)體的整個(gè)生命過程中,確保其轉(zhuǎn)錄表達(dá)和終止的方式,擾亂這一過程則可能成為改變胚胎表型的分子機(jī)制之一。胚胎發(fā)育時(shí)期,DNA甲基化狀態(tài)對(duì)環(huán)境因素尤為敏感。在“發(fā)育程序化”過程中,宮內(nèi)環(huán)境可以通過影響DNA甲基化狀態(tài),參與疾病的發(fā)生。那么,在IUGR大鼠腎臟中是否確實(shí)存在DNA甲基化模式的異常改變,這一問題的探討將有助于更好的了解“發(fā)育程序化”的發(fā)病機(jī)制,并且在疾病發(fā)生前期尋找可能的預(yù)警標(biāo)志和治療靶標(biāo)。 在本文第一部分實(shí)驗(yàn)中,我們采用孕期全程低蛋白飲食法(6%低蛋白等熱卡飼料)建立IUGR大鼠模型,選擇新生鼠仔出生體重在正常對(duì)照組平均體重-2SD以下者為IUGR組。對(duì)照組以孕期常規(guī)飼料(含22%蛋白質(zhì))飼養(yǎng)至自然分娩,所生新生鼠仔作為正常對(duì)照組。選擇新生期、生后4周和生后12周作為研究節(jié)點(diǎn),每組每個(gè)時(shí)間點(diǎn)各取6只大鼠,Real-time PCR方法檢測(cè)腎臟WT1和IGF2mRNA水平以及甲基轉(zhuǎn)移酶DNMT1、DNMT3a和DNMT3b mRNA水平；MassARRAY(?)EpityperTM定量甲基化檢測(cè)方法檢測(cè)WT1和IGF2基因啟動(dòng)子區(qū)DNA甲基化狀態(tài)；分別于4周齡和12周齡時(shí)檢測(cè)各組大鼠24小時(shí)尿蛋白肌酐比和血肝腎功能、血脂及血糖水平；于12周齡時(shí)采用光鏡和電鏡觀察腎組織形態(tài),并在光鏡下計(jì)數(shù)腎小球數(shù)目。結(jié)果發(fā)現(xiàn),IUGR新生鼠體重明顯低于正常大鼠,腎小球數(shù)目也顯著降低。光鏡下顯示IUGR新生鼠腎臟皮質(zhì)區(qū)較薄,生腎區(qū)所占的比例較大,提示IUGR新生鼠存在明顯的腎臟發(fā)育延遲。新生期IUGR大鼠腎臟WT1的表達(dá)顯著低于正常組,而IGF2的表達(dá)則出現(xiàn)升高的趨勢(shì),定量甲基化結(jié)果顯示,此時(shí)WT1啟動(dòng)子區(qū)的甲基化水平顯著升高,與其mRNA的表達(dá)水平呈明顯相關(guān)性。而IGF2基因的甲基化水平則未出現(xiàn)顯著的變化。同時(shí)我們也看到新生期DNMT1和DNMT3a的表達(dá)明顯升高,并與WT1基因DNA甲基化水平呈線性相關(guān)。因此我們推測(cè),WT1的降低可能直接或通過減弱對(duì)IGF2的抑制使其表達(dá)升高間接導(dǎo)致輸尿管芽分支異常,影響后腎胚芽的分化,并最終導(dǎo)致腎單位減少。而]3NMT1和DNMT3a調(diào)節(jié)的WT1啟動(dòng)子區(qū)甲基化水平的增加是WT1表達(dá)減少的重要機(jī)制。當(dāng)IUGR大鼠進(jìn)入成年期后,腎臟WT1和IGF2的表達(dá)顯著高于正常組,且該組大鼠出現(xiàn)了明顯的腎功能異常,主要表現(xiàn)為尿蛋白肌酐比顯著增高,并伴有尿素氮、尿酸和胱抑素-C的增高。電鏡下還可觀察到其足細(xì)胞出現(xiàn)了明顯的足突融合。定量甲基化分析顯示,WT1啟動(dòng)子區(qū)甲基化水平顯著降低,并與其mRNA的表達(dá)水平呈明顯相關(guān)性,DNMT1和DNMT3b也出現(xiàn)了明顯降低。但與新生期相同,IGF2基因啟動(dòng)子區(qū)甲基化狀態(tài)依然沒有明顯的改變。 以往認(rèn)為,IUGR兒童肌肉力量不足,能量需求高,為達(dá)到生長(zhǎng)追趕,應(yīng)給予高蛋白飲食,但快速的生長(zhǎng)追趕也可能增加發(fā)生成年期代謝疾病的風(fēng)險(xiǎn),如肥胖等。我科課題組有關(guān)IUGR新生仔鼠生后高蛋白飲食干預(yù)的研究發(fā)現(xiàn),高蛋白飲食不僅不能糾正生后腎小球數(shù)目的減少,反而加重高血壓和蛋白尿的嚴(yán)重程度。因此,有人提出,生后飲食限制可降低肥胖的發(fā)生率。研究表明,IUGR大鼠生后低蛋白飲食可增加其糖耐量和胰島素敏感性。那么,生后蛋白限制,以維持其與宮內(nèi)環(huán)境一致,會(huì)不會(huì)改善IUGR胎兒的腎臟損傷情況?還缺乏相關(guān)的研究報(bào)道。目前有關(guān)IUGR胎兒生后的合理營(yíng)養(yǎng)干預(yù)及營(yíng)養(yǎng)干預(yù)對(duì)腎臟的影響和可能的影響機(jī)制仍未完全闡明。 為探討IUGR大鼠生后不同的營(yíng)養(yǎng)干預(yù)對(duì)其不同發(fā)育階段腎臟WT1和IGF2基因DNA甲基化狀態(tài)的影響及其與腎臟功能的關(guān)系,我們進(jìn)行了第二部分的研究。對(duì)IUGR新生鼠生后分別給予常規(guī)飼料(含22%蛋白質(zhì),IUGR+N組)、高蛋白飼料(含30%蛋白質(zhì),IUGR+H組)和低蛋白飼料(含6%蛋白質(zhì),IUGR+L組)喂養(yǎng)直至12周齡,同時(shí)設(shè)立正常對(duì)照組(孕期常規(guī)飼料飼養(yǎng)至自然分娩,所生新生鼠仔生后給予常規(guī)飼料喂養(yǎng)直至生后12周齡)。與第一部分同樣的方法檢測(cè)各組大鼠新生期、4周齡和12周齡腎臟WT1和IGF2mRNA水平,分析這兩個(gè)基因啟動(dòng)子區(qū)DNA甲基化水平,以及DNMT1、DNMT3a和DNMT3b mRNA水平,同時(shí)檢測(cè)4周齡和12周齡24小時(shí)尿蛋白肌酐比、血肝腎功能、血脂和血糖等生化指標(biāo)。結(jié)果顯示,高蛋白喂養(yǎng)的大鼠體重增長(zhǎng)并不理想,雖然第4周也出現(xiàn)了生長(zhǎng)追趕,但至成年期體重始終低于對(duì)照組和生后正常喂養(yǎng)組。同時(shí)我們發(fā)現(xiàn),高蛋白喂養(yǎng)大鼠4周齡時(shí)即出現(xiàn)尿蛋白肌酐比明顯升高,至大鼠12周齡時(shí),其尿蛋白肌酐比仍然高于其他各組,并且血尿素氮和尿酸水平已顯著升高。而低蛋白喂養(yǎng)組大鼠生長(zhǎng)發(fā)育明顯受限,生后沒有出現(xiàn)生長(zhǎng)追趕,雖然其血總蛋白和白蛋白水平明顯低于其他各組,但尿蛋白肌酐比及腎功能情況卻與對(duì)照組無顯著差異,優(yōu)于生后正常喂養(yǎng)和高蛋白喂養(yǎng)組。Real-time PCR結(jié)果顯示,至成年期兩組營(yíng)養(yǎng)干預(yù)大鼠WT1和IGF2的表達(dá)均較對(duì)照組出現(xiàn)了明顯的增高,其中,低蛋白組IGF2表達(dá)的增加十分顯著。與第一部分不同的是,此時(shí)兩種不同的營(yíng)養(yǎng)干預(yù)組腎臟WT1和IGF2基因的甲基化水平均顯著降低,這種變化從4周齡一直持續(xù)到12周齡,并且在12周齡時(shí),兩組甲基化水平已顯著低于生后正常喂養(yǎng)組,這與WT1和IGF2mRNA的高表達(dá)呈明顯的線性關(guān)系。高蛋白喂養(yǎng)大鼠腎臟I)NMT1在12周齡時(shí)表達(dá)顯著降低,與該時(shí)期兩個(gè)基因的低甲基化狀態(tài)呈顯著相關(guān)性,而三種甲基轉(zhuǎn)移酶在低蛋白組大鼠腎臟都沒有出現(xiàn)明顯改變。 綜上所述,不良的宮內(nèi)環(huán)境可影響WT1的甲基化狀態(tài),繼而導(dǎo)致其異常表達(dá),而不同發(fā)育階段WT1和IGF2的異常表達(dá)可能參與了IUGR大鼠腎小球的減少和成年期蛋白尿的發(fā)生。IUGR大鼠生后高蛋白喂養(yǎng)可通過改變啟動(dòng)子區(qū)甲基化狀態(tài)調(diào)節(jié)WT1和IGF2的異常表達(dá),從而造成腎臟損傷；而生后保持宮內(nèi)環(huán)境一致的蛋白喂養(yǎng)雖能減輕腎臟負(fù)擔(dān),亦可增加通過啟動(dòng)子區(qū)甲基化水平調(diào)節(jié)WT1和IGF2異常表達(dá),增加腎臟損傷風(fēng)險(xiǎn),且不利于生長(zhǎng)發(fā)育。因而,我們應(yīng)在現(xiàn)有研究基礎(chǔ)上繼續(xù)探索新的營(yíng)養(yǎng)干預(yù)方式使IUGR胎兒既保證生長(zhǎng)發(fā)育又不增加腎臟負(fù)擔(dān),從而更好的保護(hù)腎功能。
[Abstract]:Various studies show that intrauterine malnutrition can continuously change the structure , function and metabolism of the organism , cause intrauterine growth retardation ( IUGR ) , and may be an important risk factor for the development of hypertension , coronary heart disease , insulin resistance , type 2 diabetes , obesity and chronic kidney disease .
Long - term follow - up showed that the incidence of proteinuria and end - stage renal disease was significantly lower in IUGR group than in the normal group , and the incidence of proteinuria and hypertension was significantly increased in IUGR group . However , the exact molecular mechanism of " developmental programmed " kidney disease was still not clarified .
The study reported that Wilms tumor 1 gene ( WT1 ) and insulin - like growth factor 2 ( IGF2 ) play an important role in the development of kidney . In the course of development , WT1 and IGF2 may be the key elements in the development of kidney disease . In the development process , the expression and termination of WT1 gene may be caused by the expression of WT1 and IGF2 .
In the first part of the experiment , the IUGR rat model was established by the whole course low - protein diet ( 6 % low protein , etc . ) .
The DNA methylation status of WT1 and IGF2 gene promoter regions was detected by MassARRAY ( ? ) scanning yperTM quantitative methylation detection method ;
24 - hour urinary protein creatinine ratio and blood - liver - kidney function , blood lipid and blood sugar level were measured at 4 - week and 12 - week - old respectively .
The expression of WT1 and DNMT3a in IUGR rats was significantly lower than that in normal rats . The results showed that the expression of WT1 and DNMT3a in IUGR rats was significantly higher than that in normal rats .
In the past , IUGR children ' s muscle strength is insufficient and energy demand is high . In order to achieve the growth - catching effect , high - protein diet should be administered , but rapid growth - catching may increase the risk of adult metabolic diseases , such as obesity , etc . The study shows that the high - protein diet can not only correct the decrease of postnatal glomerulus , but also increase the severity of hypertension and proteinuria .
In order to investigate the effects of different nutritional interventions on the methylation status of WT1 and IGF2 gene in different developmental stages of IUGR rats and their relationship with renal function , we carried out the study of the second part . For IUGR newborn rats , we fed the conventional feed ( including 22 % protein , IUGR + H group ) and low protein feed ( including 6 % protein , IUGR + L group ) and low protein feed ( including 6 % protein , IUGR + L group ) until 12 weeks of age . The levels of WT1 and IGF2 mRNA in the kidney of rats were significantly lower than those in the control group , but the levels of serum total protein and serum uric acid were significantly lower than those in the control group .
In conclusion , the abnormal expression of WT1 and IGF2 in IUGR rats may affect the methylation status of WT1 and the abnormal expression of WT1 and IGF2 in IUGR rats . The abnormal expression of WT1 and IGF2 can be regulated by changing methylation status of the promoter region , resulting in kidney injury .
Therefore , we should continue to explore new nutrition intervention methods to make IUGR fetus grow and develop without increasing the kidney burden , so as to better protect renal function .
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R725
本文編號(hào)：2125433
[Abstract]:Various studies show that intrauterine malnutrition can continuously change the structure , function and metabolism of the organism , cause intrauterine growth retardation ( IUGR ) , and may be an important risk factor for the development of hypertension , coronary heart disease , insulin resistance , type 2 diabetes , obesity and chronic kidney disease .
Long - term follow - up showed that the incidence of proteinuria and end - stage renal disease was significantly lower in IUGR group than in the normal group , and the incidence of proteinuria and hypertension was significantly increased in IUGR group . However , the exact molecular mechanism of " developmental programmed " kidney disease was still not clarified .
The study reported that Wilms tumor 1 gene ( WT1 ) and insulin - like growth factor 2 ( IGF2 ) play an important role in the development of kidney . In the course of development , WT1 and IGF2 may be the key elements in the development of kidney disease . In the development process , the expression and termination of WT1 gene may be caused by the expression of WT1 and IGF2 .
In the first part of the experiment , the IUGR rat model was established by the whole course low - protein diet ( 6 % low protein , etc . ) .
The DNA methylation status of WT1 and IGF2 gene promoter regions was detected by MassARRAY ( ? ) scanning yperTM quantitative methylation detection method ;
24 - hour urinary protein creatinine ratio and blood - liver - kidney function , blood lipid and blood sugar level were measured at 4 - week and 12 - week - old respectively .
The expression of WT1 and DNMT3a in IUGR rats was significantly lower than that in normal rats . The results showed that the expression of WT1 and DNMT3a in IUGR rats was significantly higher than that in normal rats .
In the past , IUGR children ' s muscle strength is insufficient and energy demand is high . In order to achieve the growth - catching effect , high - protein diet should be administered , but rapid growth - catching may increase the risk of adult metabolic diseases , such as obesity , etc . The study shows that the high - protein diet can not only correct the decrease of postnatal glomerulus , but also increase the severity of hypertension and proteinuria .
In order to investigate the effects of different nutritional interventions on the methylation status of WT1 and IGF2 gene in different developmental stages of IUGR rats and their relationship with renal function , we carried out the study of the second part . For IUGR newborn rats , we fed the conventional feed ( including 22 % protein , IUGR + H group ) and low protein feed ( including 6 % protein , IUGR + L group ) and low protein feed ( including 6 % protein , IUGR + L group ) until 12 weeks of age . The levels of WT1 and IGF2 mRNA in the kidney of rats were significantly lower than those in the control group , but the levels of serum total protein and serum uric acid were significantly lower than those in the control group .
In conclusion , the abnormal expression of WT1 and IGF2 in IUGR rats may affect the methylation status of WT1 and the abnormal expression of WT1 and IGF2 in IUGR rats . The abnormal expression of WT1 and IGF2 can be regulated by changing methylation status of the promoter region , resulting in kidney injury .
Therefore , we should continue to explore new nutrition intervention methods to make IUGR fetus grow and develop without increasing the kidney burden , so as to better protect renal function .
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R725
【參考文獻(xiàn)】
中國(guó)期刊全文數(shù)據(jù)庫 前1條
1 林其德;胎兒宮內(nèi)發(fā)育遲緩的定義及分類[J];中國(guó)實(shí)用婦科與產(chǎn)科雜志;2002年01期
，本文編號(hào)：2125433
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