本文選題：叉頭狀轉(zhuǎn)錄因子O1 + 糖尿病腎病�。� 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：背景糖尿病腎病(diabetic nephropathy,DN)是終末期腎病最常見的病因。臨床腎病最早的表現(xiàn)是出現(xiàn)持續(xù)微量白蛋白尿,繼而出現(xiàn)持續(xù)性蛋白尿,進展為顯性糖尿病腎病。隨后,腎小球濾過率(GFR)逐漸下降,5年內(nèi),50%患者發(fā)展為終末期腎臟疾病。雖然過去糖尿病腎病被認(rèn)為是腎小球疾病為主的病變,現(xiàn)今越來越多的證據(jù)表明,腎功能降低程度與腎小管間質(zhì)病變程度相關(guān)性更高。雖然多數(shù)糖尿病腎病患者最初是由于腎小球改變導(dǎo)致蛋白尿,腎間質(zhì)改變在糖尿病腎病長期病變中也發(fā)揮了重要作用。隨著越來越多研究關(guān)注病理性間質(zhì)改變,主要集中在細(xì)胞在纖維化發(fā)生中的作用,如近端小管上皮細(xì)胞(PTC)或間質(zhì)細(xì)胞。因此,研究高糖環(huán)境下PTC功能改變,以及PTC在糖尿病腎病腎間質(zhì)纖維化中的作用具有重要意義。叉頭框轉(zhuǎn)錄因子O1(FoxO1)是一類重要的調(diào)節(jié)因子,主要調(diào)控葡萄糖和脂質(zhì)代謝、氧化應(yīng)激反應(yīng)與氧化還原信號、細(xì)胞周期和細(xì)胞凋亡等方面。本課題組前期研究發(fā)現(xiàn),上調(diào)DN大鼠腎皮質(zhì)中FoxO1,可緩解腎小球病變及腎小管病變,包括系膜細(xì)胞、足細(xì)胞和腎小管上皮細(xì)胞損傷。硫氧還蛋白相互作用蛋白(TXNIP),也被稱為維生素D3上調(diào)蛋白-1(VDUP-1)或硫氧還蛋白結(jié)合蛋白2(TBP-2),是硫氧還蛋白(Trx)的內(nèi)源抑制劑,在調(diào)節(jié)血糖和血脂代謝、炎癥反應(yīng)、糖尿病等均有重要作用。Trx-Txnip系統(tǒng)對維持細(xì)胞氧化還原狀態(tài)非常重要。有研究顯示,高糖狀態(tài)下系膜細(xì)胞中,VDUP-1的表達迅速升高,IV型膠原α1鏈(COL4A1)mRNA升高,IV型膠原蛋白積累。基因芯片分析顯示,高糖環(huán)境下人近端小管細(xì)胞系(HK-2細(xì)胞),硫氧還蛋白相互作用蛋白(TXNIP)上調(diào)明顯。因此,本研究推測,在腎小管中,FoxO1可能通過抑制TXNIP,減輕高糖誘導(dǎo)的ROS升高,進而緩解糖尿病腎病腎小管氧化損傷和間質(zhì)纖維化。目的研究FoxO1/Txnip對糖尿病腎病近端小管氧化損傷和間質(zhì)纖維化的影響,并對其機制進行探討。方法體外實驗中,應(yīng)用CRISPR/CAS 9技術(shù)構(gòu)建FoxO1敲除(KO)及過表達(KI)穩(wěn)轉(zhuǎn)HK-2細(xì)胞株,并在FoxO1敲除細(xì)胞株中轉(zhuǎn)染Txnip siRNA。分組為正常糖濃度組(NG組)、高糖(25mmol/L)組(HG組),高糖+FoxO1敲除組(KO組)、高糖+FoxO1過表達組(KI組)、高糖+FoxO1敲除+Txnip siRNA組(KO+Txnip siRNA組)以及高糖+FoxO1敲除+陰性對照組(NC組)。實時熒光定量PCR(Real-time PCR)以及Western blotting檢測各組FoxO1、pFoxO1、Txnip、Trx、FN、ColⅣ等mRNA和蛋白的表達水平;流式細(xì)胞儀檢測ROS水平等相關(guān)氧化應(yīng)激指標(biāo)。體內(nèi)實驗中,應(yīng)用受精卵顯微注射構(gòu)建腎臟特異性過表達FoxO1轉(zhuǎn)基因小鼠,分組:正常小鼠組(NG組)、糖尿病組(DM組)、轉(zhuǎn)基因正常組(FoxO1-Tg組)、轉(zhuǎn)基因糖尿病組(FoxO1-Tg DM組)。12周末心臟抽血檢測血清肌酐、尿素氮;留取24小時尿檢測24小時尿總蛋白,HE染色、Masson染色、PAS染色及透射電鏡觀察腎臟病理學(xué)變化情況;取腎組織行實時熒光定量PCR(Real-time PCR)以及Western blotting檢測各組FoxO1、pFoxO1、Txnip、Trx、FN、ColⅣ等mRNA和蛋白的表達水平;免疫組化檢測FN、ColⅣ等蛋白表達水平。結(jié)果1.各組FoxO1表達及活性改變:與NG組相比,HG組Fox O1 mRNA及蛋白水平無明顯變化(P0.05),p-FoxO1/FoxO1的比值增高(P0.05),表明高糖對FoxO1轉(zhuǎn)錄活性有抑制作用;與HG組相比,KI組FoxO1 mRNA及蛋白水平均升高,p-FoxO1/FoxO1的比值增大(P0.05)。2.各組細(xì)胞中FoxO1對Txnip-Trx和ROS的影響:與NG組相比,HG組Txnip mRNA及蛋白表達均增多,Trx表達減少,ROS水平升高(均P0.05)。與HG組相比,KI組Txnip mRNA及蛋白表達均減少,Trx表達增多,ROS水平降低(均P0.05);KO組中Txnip mRNA及蛋白表達明顯增多,Trx表達明顯減少,ROS明顯升高(均P0.05)。而與KO組相比,KO+Txnip siRNA組Txnip mRNA及蛋白表達均降低,Trx表達增多,ROS水平有所降低(均P0.05)。3.FoxO1對腎間質(zhì)纖維化的影響:HG組與NG組相比,FN及Col mRⅣNA及蛋白表達均升高(均P0.05)。與HG組相比,KI組FN和ColⅣmRNA及蛋白表達降低(均P0.05);KO組FN和ColⅣmRNA及蛋白表達增多(均P0.05)。與KO組相比,KO+Txnip siRNA組FN及ColⅣmRNA和蛋白表達明顯降低(均P0.05)。4.各組小鼠腎小管中FoxO1表達情況:與NG組相比,DM組小鼠腎臟Fox O1mRNA和蛋白表達無變化(均P0.05),p-FoxO1/FoxO1比值明顯升高(均P0.05)。與DM組相比,FoxO-Tg DM組中p-FoxO1/FoxO1比值明顯降低(均P0.05)。與NG和DM組小鼠相比,FoxO1-Tg組和FoxO1-Tg DM組小鼠腎臟FoxO1 mRNA及蛋白表達增多(均P0.05)。5.各組小鼠腎小管中FoxO1抑制TXNIP信號通路的激活:與NG組和FoxO1-Tg組相比,DM組與FoxO1-Tg DM組Txnip mRNA和蛋白水平升高,Trx表達減少(均P0.05)。與DM組相比,FoxO1-Tg DM組Txnip mRNA和蛋白水平相對降低,Trx表達增多(均P0.05)。6.FoxO1緩解糖尿病腎病小鼠腎小管間質(zhì)纖維化:與NG組相比,DM組FN和Col mRⅣNA及蛋白表達均升高(均P0.05),表明糖尿病腎病時,小鼠腎臟發(fā)生間質(zhì)纖維化。與DM組相比,FoxO1-Tg DM組FN和ColⅣ表達明顯降低(均P0.05)。7.FoxO1可以有效緩解糖尿病小鼠腎臟損傷:與NG組相比,DM組體重明顯降低,腎重比明顯升高,血糖水平明顯升高,24h尿蛋白及血肌酐、尿素氮水平均明顯升高(均P0.05)。與DM組相比,FoxO1-Tg DM組體重明顯增加,腎重比明顯降低(均P0.05),血糖未見明顯變化(P0.05),24h尿蛋白及血肌酐、尿素氮水平均明顯降低(均P0.05)。與正常組相比,DM組小鼠腎小管細(xì)胞萎縮,基底膜異常增厚,膠原纖維增多,間質(zhì)纖維化明顯;與DM組相比,FoxO1-Tg DM組病變較輕,基膜均勻,稍增厚,膠原纖維少,間質(zhì)纖維化程度較輕。結(jié)論1.高糖能降低腎小管中FoxO1活性,誘導(dǎo)氧化損傷和間質(zhì)纖維化。2.過表達FoxO1明顯改善糖尿病小鼠蛋白尿和間質(zhì)纖維化程度,其機制可能與抑制TXNIP/Trx/ROS通路有關(guān)。
[Abstract]:Background diabetic nephropathy (diabetic nephropathy, DN) is the most common cause of end-stage renal disease. The earliest manifestation of clinical nephropathy is the emergence of persistent microalbuminuria, followed by persistent proteinuria and progressive diabetic nephropathy. Then, the glomerular filtration rate (GFR) decreases gradually, and within 5 years, 50% patients develop to end-stage renal disease. Although in the past, diabetic nephropathy is considered as a major glomerular disease, there is growing evidence that the degree of renal dysfunction is more associated with the degree of renal tubulointerstitial lesions. Although most diabetic nephropathy patients are initially due to glomerular changes in proteinuria, renal interstitial changes are in the long-term pathological changes of diabetic nephropathy. It also plays an important role. As more and more attention is paid to pathological interstitial changes, it is mainly focused on the role of cells in the pathogenesis of fibrosis, such as proximal tubular epithelial cells (PTC) or interstitial cells. Therefore, it is important to study the changes of PTC function in high glucose environment and the role of PTC in the renal interstitial fibrosis of diabetic nephropathy. The forked frame transcription factor O1 (FoxO1) is an important regulation factor, which mainly regulates glucose and lipid metabolism, oxidative stress reaction and redox signal, cell cycle and cell apoptosis. Earlier study in our group found that up regulation of FoxO1 in renal cortex of DN rats could relieve glomerular lesions and renal tubular lesions, including mesangial cells. Thioredoxin protein interaction protein (TXNIP), also known as vitamin D3 up-regulated protein -1 (VDUP-1) or thioredoxin binding protein 2 (TBP-2), is an endogenous inhibitor of thioredoxin (Trx). It has an important role in regulating blood glucose and blood lipid metabolism, inflammatory response, and diabetes, and so on. It is very important to maintain the redox state of cells. Studies have shown that the expression of VDUP-1 in mesangial cells of high glucose state increases rapidly, IV collagen alpha 1 chain (COL4A1) mRNA increases and IV collagen accumulation. Gene chip analysis shows that human proximal tubule cell line (HK-2 cell) and thioredoxin protein interaction protein (TXNIP) in high glucose environment Therefore, this study suggests that in renal tubules, FoxO1 may alleviate the oxidative damage of renal tubules and interstitial fibrosis in diabetic nephropathy by inhibiting TXNIP and alleviating the increase of ROS induced by high glucose. Objective to study the effect of FoxO1/Txnip on the oxidative damage and interstitial fibrosis of proximal tubules in diabetic nephropathy and to explore its mechanism. Methods in vitro, CRISPR/CAS 9 technique was used to construct FoxO1 knockout (KO) and overexpression (KI) to stabilize HK-2 cell lines, and transfected Txnip siRNA. into normal glucose concentration group (NG group), high sugar (25mmol/L) group (HG group), high sugar +FoxO1 knockout group, high sugar overexpression group, high sugar overexpression group, and high glucose knocking in FoxO1 knockout cell lines. SiRNA group (group KO+Txnip siRNA) and high glucose +FoxO1 knockout + negative control group (NC group). Real time fluorescent quantitative PCR (Real-time PCR) and Western blotting were used to detect the expression level of each group and protein; flow cytometry was used to detect the levels of related oxidative stress. In vivo experiments, the fertilized eggs were applied. Renal specific overexpression FoxO1 transgenic mice were microinjected into normal mice (group NG), diabetes group (group DM), transgenic normal group (group FoxO1-Tg), and genetically modified diabetes group (group FoxO1-Tg DM).12 weekend cardiac blood test for serum creatinine and urinary nitrogen; 24 hour urine test was left for 24 hours urinary total protein, HE staining, Masson staining, P. The pathological changes of kidney were observed by AS staining and transmission electron microscopy, and the expression of FoxO1, pFoxO1, Txnip, Trx, Trx, FN, Col IV and other proteins were detected by real-time fluorescence quantitative PCR (Real-time PCR) and Western blotting in the renal tissue. Compared with group NG, the level of Fox O1 mRNA and protein in group HG had no obvious changes (P0.05), and the ratio of p-FoxO1/FoxO1 increased (P0.05), indicating that high sugar had a inhibitory effect on the transcriptional activity of FoxO1. Compared with the HG group, the expression of Txnip mRNA and protein increased, the expression of Trx decreased, and the level of ROS increased (P0.05). Compared with the HG group, the Txnip mRNA and protein expression in KI group decreased, Trx expression increased and ROS level decreased. The expression of Txnip mRNA and protein in group O+Txnip siRNA decreased, the expression of Trx increased, and the level of ROS decreased (P0.05).3.FoxO1's effect on renal interstitial fibrosis: HG group and NG group were higher than NG group. The expression of protein was increased (all P0.05). Compared with the KO group, the expression of FN and Col IV mRNA and protein in the KO+Txnip siRNA group decreased significantly (all P0.05) in the renal tubules of each group of.4. mice, and the expression of the kidneys and proteins in the mice of the DM group was significantly higher than that in the NG group. The p-FoxO1/FoxO1 ratio in the DM group was significantly lower (P0.05). Compared with the NG and DM mice, the FoxO1 mRNA and protein expression in the FoxO1-Tg and FoxO1-Tg DM mice increased (P0.05) in the renal tubules of the mice. Trx expression decreased (P0.05). Compared with group DM, Txnip mRNA and protein level in FoxO1-Tg DM group decreased and Trx expression increased (all P0.05).6.FoxO1 alleviated renal tubulointerstitial fibrosis in diabetic nephropathy mice. Interstitial fibrosis. Compared with the DM group, the expression of FN and Col IV in the FoxO1-Tg DM group decreased significantly (all P0.05).7.FoxO1 could effectively alleviate the renal injury in diabetic mice. Compared with the NG group, the weight of the DM group was significantly reduced, the renal weight ratio was significantly elevated, the blood glucose level was significantly elevated, the 24h urine protein and serum creatinine, and the urea nitrogen water increased significantly (P0.05). And DM group. The weight of FoxO1-Tg DM group was significantly increased, the renal weight ratio was significantly decreased (P0.05), blood sugar was not significantly changed (P0.05), 24h urine protein and blood creatinine, and urea nitrogen water decreased significantly (P0.05). Compared with the normal group, the renal tubule cells in the DM group were atrophied, the basement membrane was thickened, the collagen fibers increased and the interstitial fibrosis was obvious; compared with the DM group, it was compared with the DM group. The pathological changes of FoxO1-Tg DM group are lighter, the basement membrane is homogeneous, the thickness of the collagen fiber is slightly thicker, the collagen fiber is less and the degree of interstitial fibrosis is lighter. Conclusion 1. high glucose can reduce the activity of FoxO1 in the renal tubules. The induced oxidative damage and the overexpression of.2. over the interstitial fibrosis can obviously improve the proteinuria and the degree of interstitial fibrosis in diabetic mice. The mechanism may be related to the inhibition of the TXNIP/Trx/ROS pathway. Of

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.2;R692.9

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1 王曉杰;組蛋白去乙�；�4特異性促進糖尿病腎病足細(xì)胞損傷[D];山東大學(xué);2015年

2 張永;MiR-346在抗TGF-β信號途徑介導(dǎo)的糖尿病腎病發(fā)生和發(fā)展中的作用機制[D];武漢大學(xué);2015年

3 魏鳳江;高尿酸血癥、2型糖尿病及糖尿病微血管病變的群體遺傳學(xué)研究[D];天津醫(yī)科大學(xué);2015年

4 孫士杰;胱抑素C對2型糖尿病視網(wǎng)膜病變預(yù)測價值研究[D];山東大學(xué);2015年

5 龍泓竹;益氣養(yǎng)陰通絡(luò)散結(jié)方防治早期糖尿病腎病的臨床及實驗研究[D];北京中醫(yī)藥大學(xué);2016年

6 姜e，

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