本文選題：高糖 + 足細(xì)胞��； 參考：《武漢大學(xué)》2014年博士論文

【摘要】：背景慢性腎臟病(chronic kidney disease,CKD)是嚴(yán)重威脅人民身體健康且消耗巨大社會資源的常見疾病,最近的調(diào)查顯示國內(nèi)CKD患病率約為10.8%,由于其致病因素廣泛,其發(fā)病率一直居高不下,且有逐年上升的趨勢,其高死亡率、高致殘率、高社會成本支出、低知曉率也同樣令人關(guān)注。糖尿病腎病是慢性腎臟病的主要病因之一,在全球有約3億糖尿病病人,其中約30%發(fā)展為糖尿病腎病,恩施地區(qū)也屬于糖尿病高發(fā)區(qū),根據(jù)恩施土家族苗族自治州血液凈化統(tǒng)計資料,透析病人中糖尿病腎病占14.9%。腎臟血流動力學(xué)變化、腎臟微血管病變、小球系膜細(xì)胞及基質(zhì)增生等曾被認(rèn)為是糖尿病腎病的主要病理生理變化,近年隨著對足細(xì)胞的深入研究,認(rèn)為足細(xì)胞損傷、足細(xì)胞功能改變、足細(xì)胞過度凋亡與糖尿病腎病的蛋白尿、腎小球硬化密切相關(guān),足細(xì)胞受損是糖尿病腎病發(fā)展的關(guān)鍵,因此有學(xué)者認(rèn)為糖尿病腎病的本質(zhì)就是足細(xì)胞病。 腎素血管緊張素系統(tǒng)(RAS)的激活與糖尿病腎病的發(fā)生與發(fā)展關(guān)系密切,在RAS與糖尿病腎病足細(xì)胞受損之間的復(fù)雜網(wǎng)絡(luò)關(guān)系中,PI3K-AKT-mTOR信號傳導(dǎo)途徑起著重要作用,mTOR的活化能減少足細(xì)胞自噬,促進(jìn)足細(xì)胞凋亡。雖然糖尿病腎病時觀察到了足細(xì)胞mTOR信號通路的活化及足細(xì)胞損傷,以及阻斷mTOR信號系統(tǒng)帶來的益處,但足細(xì)胞mTOR信號通路具體調(diào)控過程還在探索中,糖尿病時高糖能否直接活化足細(xì)胞mTOR信號通路、導(dǎo)致足細(xì)胞凋亡目前也不清楚。腎臟局部的RAS系統(tǒng)除了通過影響腎臟的血流動力學(xué)外,還影響尿蛋白排泄、系膜基質(zhì)增生、小管間質(zhì)纖維化,而足細(xì)胞作為糖尿病腎病病變的核心,不僅是RAS引起損傷作用的靶目標(biāo),亦是局部RAS的重要來源,RAS激活對足細(xì)胞本身的影響目前尚未完全明了,特別是對足細(xì)胞凋亡、足細(xì)胞mTOR信號通路的影響研究更少。 目的觀察高糖誘導(dǎo)足細(xì)胞mTOR表達(dá)及凋亡改變以及腎素抑制劑對其的影響。 方法1.將培養(yǎng)的成熟足細(xì)胞分為對照組、高滲組、高糖組,對照組置于含5mmol/L葡萄糖的RPMI1640培養(yǎng)液,高滲組為含5mmol/L葡萄糖、25mmol/L甘露醇的RPMI1640培養(yǎng)液,高糖組為含30mmol/L葡萄糖的RPMI1640培養(yǎng)液。各組足細(xì)胞培養(yǎng)24小時后應(yīng)用RT-PCR測定各組mTOR mRNA表達(dá),Western blot測定各組足細(xì)胞mTOR蛋白表達(dá),TUNEL法測定各組足細(xì)胞凋亡率。2.將培養(yǎng)足細(xì)胞分為對照組、無義轉(zhuǎn)染組、siRNA轉(zhuǎn)染組,轉(zhuǎn)染組加入轉(zhuǎn)染試劑(Lipofecter旨質(zhì)體)、mTOR siRNA進(jìn)行轉(zhuǎn)染,無義對照組加入轉(zhuǎn)染試劑、對照(無義)siRNA,空白對照組僅加轉(zhuǎn)染試劑(空脂質(zhì)體)。按轉(zhuǎn)染試劑盒說明書轉(zhuǎn)染24小時,將轉(zhuǎn)染成功后的足細(xì)胞置于含30mmol/L葡萄糖RPMI1640培養(yǎng)液繼續(xù)培養(yǎng)24小時。通過RT-PCR測定各組mTOR mRNA表達(dá),Western blot測定各組足細(xì)胞mTOR蛋白表達(dá),TUNEL法測定各組足細(xì)胞凋亡率。3.將培養(yǎng)的足細(xì)胞分為對照組、低劑量Aliskiren組(含10nmol/L的Aliskiren)、中劑量Aliskiren組(含20nmol/L的Aliskiren)、高劑量Aliskiren組(含30nmol/L的Aliskiren),在30mmol/L葡萄糖RPMI1640培養(yǎng)液培養(yǎng)24小時后應(yīng)用RT-PCR測定各組mTOR mRNA表達(dá),Western blot測定各組足細(xì)胞mTOR蛋白表達(dá),TUNEL法測定各組足細(xì)胞凋亡。 結(jié)果在高糖刺激下,足細(xì)胞表達(dá)mTOR mRNA較高滲組及對照組顯著上調(diào)(P0.01),mTOR蛋白表達(dá)也顯著上調(diào)(P0.01),足細(xì)胞凋亡高于高滲組及對照組(P0.01)。應(yīng)用siRNA技術(shù)沉默mTOR基因表達(dá)后,足細(xì)胞mTOR mRNA及mTOR蛋白均較對照組和無義轉(zhuǎn)染組顯著下調(diào)(P0.01),轉(zhuǎn)染組在高糖刺激下足細(xì)胞凋亡率顯著下降(P0.05)。給予腎素抑制劑Aliskiren干預(yù)后,足細(xì)胞mTOR蛋白表達(dá)較對照組下調(diào)(P0.01),足細(xì)胞凋亡也較對照組下降(P0.01),隨著干預(yù)劑量的增加,其mTOR蛋白表達(dá)下調(diào)更加顯著,三組劑量組間有統(tǒng)計學(xué)差異(P0.05)。 結(jié)論高糖可能通過上調(diào)足細(xì)胞mTOR表達(dá)誘導(dǎo)其凋亡；腎素抑制劑對足細(xì)胞的保護(hù)作用可能與其下調(diào)足細(xì)胞mTOR表達(dá),減少高糖刺激下的足細(xì)胞凋亡有關(guān)。 目的觀察腎素抑制劑Aliskiren對糖尿病腎病大鼠腎臟mTOR表達(dá)、尿液足細(xì)胞排泄的影響。 方法40只Wistar大鼠采用腹腔內(nèi)一次性注射STZ制作糖尿病模型,隨機(jī)分糖尿病組及Aliskiren治療組,另20只正常Wistar大鼠作為對照組,Aliskiren治療組每日給予Aliskiren10mg/kg一次性灌胃,8周后留取尿液計數(shù)各組大鼠尿液足細(xì)胞排泄數(shù)；取大鼠腎皮質(zhì),RT-PCR測定各組大鼠腎臟mTORmRNA表達(dá),Western blot測定mTOR蛋白表達(dá)。 結(jié)果糖尿病大鼠尿液足細(xì)胞排泄顯著高于對照組(P0.01),應(yīng)用Aliskiren干預(yù)8周后,糖尿病大鼠足細(xì)胞排泄明顯減少(P0.01)；糖尿病大鼠腎組織mTOR表達(dá)顯著高于對照組(P0.01),Aliskiren治療后大鼠腎組織mTOR表達(dá)較糖尿病組顯著下調(diào)(P0.01)。 結(jié)論腎素抑制劑能減少糖尿病大鼠足細(xì)胞排泄,減少尿蛋白,其腎臟保護(hù)機(jī)制可能與下調(diào)腎組織mTOR表達(dá)有關(guān)。
[Abstract]:Chronic kidney disease (CKD) is a common disease which seriously threatens the health of the people and consumes huge social resources. The recent survey shows that the prevalence rate of CKD in China is about 10.8%. Because of its widespread pathogenicity, the incidence of the disease has always been high, and it has a trend of increasing year by year. The high mortality rate, high disability rate, and high rate of disability are high. Diabetes nephropathy is one of the main causes of chronic kidney disease. There are about 300 million diabetic patients around the world, about 30% of them are diabetic nephropathy, and Enshi is also a high risk area for diabetes. According to the blood purification statistics of the Enshi Tujia and Miao Autonomous Prefecture, the diabetes mellitus is dialysable. Renal hemodynamic changes in 14.9%., renal microvascular lesions, glomerular mesangial cells and matrix hyperplasia have been considered as the main pathophysiological changes in diabetic nephropathy. In recent years, with the in-depth study of podocytes, podocyte injury, podocyte function change, hyperapoptosis of podocytes and proteinuria in diabetic nephropathy have been considered. Glomerular sclerosis is closely related and podocyte damage is the key to the development of diabetic nephropathy. Therefore, some scholars believe that the essence of diabetic nephropathy is podocyte.
The activation of renin angiotensin system (RAS) is closely related to the occurrence and development of diabetic nephropathy. In the complex network relationship between RAS and diabetic nephrotic foot cell damage, the PI3K-AKT-mTOR signal transduction pathway plays an important role. The activation of mTOR can reduce the autophagy of podocyte and promote the apoptosis of the podocyte. The activation of podocyte mTOR signaling pathway and podocyte damage, as well as the benefits of blocking the mTOR signal system, are also detected, but the specific regulatory process of the mTOR signaling pathway is still explored. Whether the hyperglycemic glucose can directly activate the mTOR signal pathway of the podocyte leads to the absence of the apoptosis of the podocyte. The partial RAS system in the kidney is not known. By affecting the hemodynamics of the kidney, it also affects urinary protein excretion, mesangial matrix hyperplasia and tubulointerstitial fibrosis, and podocyte as the core of diabetic nephropathy is not only the target of RAS causing damage, but also an important source of local RAS. The effect of RAS activation on the foot cell itself is not completely clear, especially There is less research on podocyte apoptosis and podocyte mTOR signaling pathway.
Objective To observe the changes of mTOR expression and apoptosis in podocytes induced by high glucose and the effects of renin inhibitors on them.
Methods 1. the cultured mature podocytes were divided into control group, hypertonic group, high glucose group and control group in RPMI1640 culture containing 5mmol/L glucose. Hypertonic group was RPMI1640 culture solution containing 5mmol/L glucose, 25mmol/L mannitol, and high sugar group was RPMI1640 culture medium containing 30mmol/L glucose. After 24 hours of culture, RT-PCR was used in each group. The expression of mTOR mRNA in each group, Western blot was used to determine the expression of mTOR protein in each group of podocytes. TUNEL method was used to determine the apoptotic rate of foot cells in each group. The podocytes were divided into control group, no sense transfection group, siRNA transfection group, the transfection group was added to the transfection reagent (Lipofecter plastid), mTOR siRNA transfected, the non sense control group was added to the transfection reagent, and the control (Wu Yi) s IRNA, the blank control group only added the transfection reagent (air liposome). After transfection of the transfection kit for 24 hours, the transfected podocyte was placed in the culture solution containing 30mmol/L glucose RPMI1640 for 24 hours. The expression of mTOR mRNA in each group was determined by RT-PCR, Western blot was used to determine the expression of mTOR protein in each group, and the TUNEL method was used to determine each group. The apoptotic rate of.3. was divided into the control group, the low dose Aliskiren group (including 10nmol/L Aliskiren), the medium dose Aliskiren group (Aliskiren containing 20nmol/L), the high dose Aliskiren group (30nmol/L Aliskiren), and the determination of the expression in the 30mmol/L glucose RPMI1640 culture 24 hours after the culture. N blot was used to detect the expression of mTOR protein in podocytes of each group, and podocyte apoptosis was detected by TUNEL.
Results under the high glucose stimulation, the expression of mTOR mRNA in the hypertonic group and the control group increased significantly (P0.01), and the expression of mTOR protein was also significantly up (P0.01). The apoptosis of the Poda was higher than that in the hypertonic group and the control group (P0.01). The mTOR mRNA and mTOR protein of the podocytes were significantly lower than those of the control group and the non sense transfection group after the siRNA technique was silent on the expression of mTOR gene. The apoptosis rate of foot cells decreased significantly (P0.05) in the transfected group under the stimulation of high glucose (P0.01). The expression of mTOR protein in the poddine was lower than that of the control group (P0.01), and the apoptosis of the Poda was also lower than that of the control group (P0.01). The expression of mTOR protein decreased significantly with the increase of intervention dose. There were three groups of dose groups. Statistical difference (P0.05).
Conclusion high glucose may induce apoptosis by up regulating the expression of mTOR in podocyte, and the protective effect of renin inhibitor on podocyte may be related to the down-regulation of mTOR expression in podocyte and the reduction of podocyte apoptosis under high glucose stimulation.
Objective To observe the effect of renin inhibitor Aliskiren on mTOR expression and urine podocyte excretion in diabetic nephropathy rats.
Methods 40 Wistar rats were treated by intraperitoneal injection of STZ to make diabetic model, randomly divided into diabetes group and Aliskiren treatment group, the other 20 normal Wistar rats as control group. Aliskiren treatment group was given Aliskiren10mg/kg once every day, after 8 weeks, the urine podocyte excretion number of rats in each group was collected; rats were collected. In the renal cortex, RT-PCR was used to detect the mTORmRNA expression in the kidneys of each group, and the expression of mTOR protein was detected by Western blot.
Results the excretion of urine podocyte in diabetic rats was significantly higher than that of the control group (P0.01). After 8 weeks of Aliskiren intervention, the excretion of podocyte in diabetic rats decreased significantly (P0.01), and the expression of mTOR in renal tissue of diabetic rats was significantly higher than that of the control group (P0.01). The expression of mTOR in renal tissue of rats was significantly lower than that of the diabetic group after Aliskiren treatment (P0.01).
Conclusion renin inhibitor can reduce podocyte excretion and decrease urinary protein in diabetic rats, and its renal protective mechanism may be related to downregulation of mTOR expression in renal tissue.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R692

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相關(guān)期刊論文 前2條

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本文編號：1855370