本文關(guān)鍵詞： 糖尿病腎病 12-脂氧化酶 P-cadherin 腎小球 蛋白尿　出處：《吉林大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：背景:糖尿病腎病(Diabetic nephropathy,DN)是糖尿病最常見的并發(fā)癥之一,是導(dǎo)致慢性腎衰竭的重要原因及死亡原因,這一趨勢在發(fā)達國家更為明顯。蛋白尿是DN的主要臨床表現(xiàn)及核心發(fā)病環(huán)節(jié),決定了病情的嚴(yán)重程度和發(fā)展速度,且成為一種獨立的心血管危險因素。探討DN蛋白尿的機制臨床意義重大。脂氧化酶(Lipoxygenase,LO)是機體內(nèi)一類重要的氧化酶,主要作用于不飽和脂肪酸。依據(jù)其作用于花生四烯酸時氧化位置分為5-、8-、12-和15-LO。12(S)-氫氧化二十碳四烯酸[12(S)-hydroxyeicosatetraenoic acid,12(S)-HETE]是12-LO氧化花生四烯酸生成的活性脂質(zhì)產(chǎn)物。研究表明,氧化應(yīng)激和炎癥反應(yīng)是12-LO和其脂質(zhì)產(chǎn)物12(S)-HETE引起蛋白尿的主要機理。足細(xì)胞裂孔蛋白如nephrin、P-cadherin和NEPH1構(gòu)成腎小球濾過的重要屏障,研究顯示DN早期相對肥大腎小球中nephrin表達減少與蛋白尿形成相關(guān)。有研究表明P-cadherin損傷可導(dǎo)致非nephrin或NEPH1依賴的蛋白尿,那么其是否與nephrin一樣在DN蛋白尿形成中發(fā)揮重要作用呢?目前尚未見到相關(guān)報道。之前的研究證實12-LO可通過影響nephrin的表達參與DN蛋白尿形成,那么其對同樣是裂孔蛋白的P-cadherin作用如何呢?目前尚未有相關(guān)報道并因此成為本研究的目的。方法:(1)將同步化的足細(xì)胞分成四組:對照組(Ctrl);SB202190組(p38MAPK抑制劑),培養(yǎng)液中加入10~(-6) mol/L SB202190;12(S)-HETE組,培養(yǎng)液中加入10~(-7) mol/L12(S)-HETE;12(S)-HETE+SB202190組,培養(yǎng)液中加入10~(-7) mol/L 12(S)-HETE和10~(-6)mol/L SB202190,24小時后收集細(xì)胞,Western blot檢測細(xì)胞內(nèi)P-cadherin表達。(2)將微型滲透泵植入Sprague Dawley大鼠皮下,持續(xù)泵入12(S)-HETE(1 mg·Kg-1·d-1),對照組泵入乙醇胺。7天后將大鼠處死收集腎臟,應(yīng)用系列過篩法分離不同大小的腎小球,應(yīng)用RT-PCR和Western blot檢測腎小球內(nèi)P-cadherin表達。(3)將野生型和12-LO基因敲除C57/BL6小鼠分成四組:野生型對照組(WT)、野生型糖尿病組(WT+STZ)、12-LO基因敲除組(LOKO)、12-LO基因敲除糖尿病組(LOKO+STZ)。小鼠腹腔內(nèi)單次大劑量注射STZ建立1型糖尿病模型,16周后處死提取腎小球,應(yīng)用競爭性RT-PCR及Western blot檢測腎小球內(nèi)P-cadherin表達,并采用Western blot檢測腎小球內(nèi)p38MAPK、ERK1/2、JNK及其磷酸化水平。監(jiān)測小鼠血糖,收集尿液用于測定24h尿白蛋白水平。(4)給予Sprague Dawley大鼠高脂飲食及小劑量STZ腹腔注射建立2型糖尿病模型。成模后隨機分為2組:DN組和CDC(Cinnamyl-3,4-dihydroxy-α-cynanocinnamate,12-LO抑制劑)組。DN組皮下注射芝麻油,CDC組皮下注射CDC 8 mg·Kg-1,每周3次。對照組給予正常飲食。檢測8周后血糖、體重、腎重,收集腎小球,Western blot、免疫熒光、免疫組化檢測腎小球內(nèi)P-cadherin表達,ELISA檢測12(S)-HETE水平。于實驗結(jié)束前留取24 h尿測尿白蛋白。結(jié)果:(1)12(S)-HETE直接刺激明顯減少大鼠腎小球內(nèi)P-cadherin m RNA及蛋白表達(P0.05),且大、小腎小球無明顯差異。(2)12(S)-HETE可減少足細(xì)胞內(nèi)P-cadherin蛋白表達(P0.05),這一作用可以部分被p38MAPK抑制劑SB202190阻斷(P0.05)。(3)與對照組比較,WT+STZ組小鼠腎小球內(nèi)P-cadherin表達明顯減少(P0.05),而敲除12-LO基因可增加LOKO+STZ組小鼠腎小球內(nèi)P-cadherin含量(P0.05)。(4)與對照組比較,WT+STZ組小鼠腎小球內(nèi)p-p38MAPK、p-ERK1/2、p-JNK水平均明顯增加(P0.05),敲除12-LO基因僅可以減少p-p38MAPK水平(P0.05),而對p-ERK1/2和p-JNK無明顯影響(P0.05)。(5)與對照組比較,DN組大鼠血糖、腎重/體重明顯增加,腎小球體積明顯增大,尿白蛋白水平明顯增加(P0.05);皮下注射CDC可降低糖尿病大鼠腎重/體重及尿白蛋白水平,緩解腎小球肥大(P0.05)。(6)與對照組比較,DN組大鼠腎小球內(nèi)12(S)-HETE含量明顯增加(P0.05),且這一趨勢在相對肥大腎小球中更明顯(P0.05),CDC可減少糖尿病大鼠腎小球內(nèi)12(S)-HETE水平(P0.05)。(7)與對照組比較,DN組腎小球內(nèi)P-cadherin m RNA和蛋白水平明顯減少(P0.05),且在大、小腎小球中無明顯差異(P0.05),皮下注射CDC可增加糖尿病大鼠腎小球內(nèi)P-cadherin含量(P0.05)。結(jié)論:(1)12-LO作用產(chǎn)物12(S)-HETE直接刺激可降低足細(xì)胞及腎小球內(nèi)P-cadherin表達。(2)DN腎小球內(nèi)P-cadherin表達降低,抑制12-LO可上調(diào)腎小球內(nèi)P-cadherin表達水平。(3)12-LO通過p38MAPK信號通路調(diào)節(jié)DN腎小球內(nèi)P-cadherin的表達。
[Abstract]:Background: diabetic nephropathy (Diabetic nephropathy DN) is one of the most common complications of diabetes, is the cause of an important cause of chronic renal failure and death in developed countries, this trend is more obvious. The urine protein is the major clinical manifestations of DN and the core pathogenesis, determines the severity of the illness and the speed of development, and to become an independent cardiovascular risk factor. To investigate the mechanism and clinical significance of DN protein in the urine. The major lipoxygenase (Lipoxygenase, LO) is an important class of enzymes in the body, mainly on unsaturated fatty acids. On the basis of its role in the four arachidonic acid oxidation when the position is divided into 5-, 8-, 12- and 15-LO.12 (S twenty) - hydroxide carbon four acid [12 (S) -hydroxyeicosatetraenoic acid 12 (S) -HETE] is the activity of lipid oxidation products of 12-LO four arachidonic acid production. Research shows that oxidative stress and inflammatory reaction is 12-LO and its lipid production 12 (S) the main mechanism of proteinuria caused by -HETE. Podocyte Slit proteins such as nephrin, P-cadherin and NEPH1 constitute an important barrier of glomerular filtration, studies show that early DN relative expression of nephrin in glomerular hypertrophy associated with a decrease in proteinuria. Studies have shown that P-cadherin damage can lead to non nephrin or NEPH1 dependent protein in urine so, whether the nephrin like in the DN play an important role in the formation of proteinuria? Has yet to see the relevant reports. Previous studies confirmed that 12-LO can be formed by the expression of nephrin DN protein in urine, then the same is how the role of P-cadherin protein in hiatus? There have been no reports and thus become the the purpose of the study. Methods: (1) the synchronized podocytes were divided into four groups: control group (Ctrl); group SB202190 (p38MAPK inhibitor), was added to the culture medium 10~ (-6) mol/L SB202190 12 (S) -HE; TE was added to the culture medium 10~ (-7) mol/L12 (S) -HETE; 12 (S) -HETE+SB202190 were added to the culture medium 10~ (-7) mol/L 12 (S) -HETE and 10~ (-6) mol/L SB202190,24 hours after collection of cells, the expression of Western blot detected P-cadherin. (2) the micro penetration pump implantation of Sprague Dawley rats subcutaneously, continuous infusion of 12 (S) -HETE (1 mg - Kg-1 - D-1), the control group pump ethanolamine.7 days after the rats were sacrificed to collect the kidney, using a series of different size separation glomerular sieve method, the expression of P-cadherin and Western using RT-PCR blot detection (3 glomeruli.) and the wild type 12-LO gene knockout C57/BL6 mice were divided into four groups: wild type control group (WT), wild type diabetic group (WT+STZ), 12-LO gene knockout group (LOKO), 12-LO gene knockout diabetic group (LOKO+STZ). The establishment of type 1 diabetic mice intraperitoneal single dose injection STZ, after 16 weeks of glomerular extraction The expression of P-cadherin, RT-PCR and Western by competitive blot detection by Western and blot in glomeruli, glomerular detection within p38MAPK, ERK1/2, JNK and its phosphorylation level. Blood glucose monitoring in mice, urine was collected for determination of 24h urinary albumin levels. (4) treated with high fat diet Sprague Dawley rats and small dose of STZ intraperitoneal injection to establish type 2 the models of diabetes. The rats were randomly divided into 2 groups: group DN and CDC (Cinnamyl-3,4-dihydroxy- alpha -cynanocinnamate, 12-LO inhibitor) group.DN group subcutaneous injection of sesame oil, CDC group subcutaneous injection of CDC 8 mg - Kg-1, 3 times a week. The control group was given normal diet. After 8 weeks of testing blood glucose, body weight, kidney weight, collect Western blot, the glomeruli, immunofluorescence, immunohistochemistry to detect the expression of P-cadherin in glomeruli, ELISA was detected in 12 (S) -HETE level. At the end of the experiment before and collected 24 h urine test urine albumin. Results: (1) 12 (S) -HETE direct stimulation significantly reduce Little rat glomerular RNA and protein expression of P-cadherin m (P0.05), and big, no obvious difference. (2) 12 small glomeruli (S) -HETE can reduce the expression of P-cadherin protein in the podocyte (P0.05), this effect can be part of p38MAPK inhibitor SB202190 (P0.05) (3) and the control group. Comparison of group WT+STZ significantly decreased the expression of P-cadherin in glomeruli of mice (P0.05), and 12-LO gene knockout mice can increase the contents of P-cadherin LOKO+STZ in glomeruli (P0.05). (4) compared with the control group, WT+STZ group of mice glomeruli p-p38MAPK, p-ERK1/2, p-JNK levels were significantly increased (P0.05), 12-LO gene knockout only can reduce the level of p-p38MAPK (P0.05), but had no obvious effect on p-ERK1/2 and p-JNK (P0.05). (5) compared with the control group, the blood glucose of rats in the DN group, kidney weight / body weight increased, glomerular volume increased significantly, the level of urinary albumin was significantly increased (P0.05); subcutaneous injection of CDC can be reduced Diabetic rat kidney weight / body weight and urinary albumin levels, alleviate glomerular hypertrophy (P0.05). (6) compared with the control group, glomerulus in rats of DN group 12 (S) -HETE were significantly increased (P0.05), and this trend was more obvious in relative glomerular hypertrophy (P0.05), CDC can reduce glomerular diabetic rats in 12 (S) -HETE (P0.05). (7) compared with the control group, the level of P-cadherin m and RNA protein in DN group decreased significantly in glomeruli (P0.05), and no significant difference in large, small glomeruli (P0.05), subcutaneous injection of CDC can increase the contents of P-cadherin in glomeruli of diabetic rats disease (P0.05). Conclusion: (1) 12-LO 12 (S) -HETE product direct stimulation can decrease the expression of P-cadherin in podocytes and glomeruli. (2) decrease the expression of P-cadherin DN in glomeruli, inhibition of 12-LO can upregulate the expression of P-cadherin in glomerular level. (3) 12-LO regulation of DN P-cadhe in glomeruli through p38MAPK signaling pathway The expression of RIN.

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

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