本文選題：活性維生素D3　切入點(diǎn)：糖尿病腎病　出處：《東南大學(xué)》2015年博士論文

【摘要】：目的糖尿病腎病是糖尿病的嚴(yán)重并發(fā)癥,是導(dǎo)致糖尿病腎病患者致殘和死亡的主要原因之一。足細(xì)胞損傷被認(rèn)為是引起糖尿病腎病蛋白尿和腎小球硬化的關(guān)鍵因素。因此,探討足細(xì)胞損傷的機(jī)制并尋找有效可行的干預(yù)足細(xì)胞損傷的藥物是解決問題的關(guān)鍵�；钚跃S生素D3對糖尿病腎病具有顯著的腎保護(hù)作用,但其機(jī)制尚未闡明。本研究通過構(gòu)建STZ誘導(dǎo)的糖尿病腎病大鼠模型及體外足細(xì)胞培養(yǎng)模型兩方面,觀察活性維生素D3對糖尿病腎病足細(xì)胞的保護(hù)作用,同時(shí)探討可能機(jī)制。方法將SD雄性大鼠隨機(jī)分為四組：正常對照組(NC組)、活性維生素D3干預(yù)組(VD組,骨化三醇0.1μg·kg-1·d-1灌胃)、糖尿病腎病組(DN組,腹腔注射STZ 58mg·kg-1)和糖尿病腎病+活性維生素D3組(DN+VD組),定期檢測血糖、體質(zhì)量,收集尿標(biāo)本,干預(yù)18w末處死動物,檢測Scr、BUN、Ca、P和24小時(shí)尿蛋白變化,PAS和MASSON染色觀察腎臟病理改變,電鏡觀察足細(xì)胞超微結(jié)構(gòu)變化。體外研究采用體外培養(yǎng)小鼠永生系足細(xì)胞,對分化的足細(xì)胞予不同的刺激,觀察1,25(OH)2D3、TRPC6抑制劑SKF-96365以及PI3K抑制劑LY294002對高糖誘導(dǎo)足細(xì)胞損傷的影響。免疫組化法檢測腎組織WT-1、Nephrin、 Podocin、Desmin、VDR和p-Akt;免疫熒光法檢測腎組織TRPC6表達(dá)以及足細(xì)胞Nephrin、Podocin、Desmin 及 TRPC6; Real-time PCR、Western Blot檢測腎組織VDR、足細(xì)胞裂孔隔膜蛋白Nephrin、Podocin、TRPC6、足細(xì)胞損傷標(biāo)志Desmin和PI3K/p-Akt信號通路相關(guān)分子表達(dá)。結(jié)果(1)體內(nèi)研究表明,與DN組相比,DN+VD組蛋白尿減低達(dá)36%,腎臟病理損傷減輕,足細(xì)胞損傷減輕,血糖、體質(zhì)量以及血尿素氮之間無差異(均P0.05)；血肌酐、鈣、磷在四組之間未見差異(均P0.05)。與NC組相比,DN組Podocin、 Nephrin表達(dá)量均明顯減低(均P0.05),足細(xì)胞損傷標(biāo)志Desmin蛋白表達(dá)量顯著增加(P0.05)。與DN組相比,DN+VD組Podocin、Nephrin表達(dá)量均明顯增加(均P0.05),足細(xì)胞損傷標(biāo)志Desmin表達(dá)量顯著減低(P0.05)。WT-1在四組之間未見統(tǒng)計(jì)學(xué)差異。體外研究也證實(shí),與對照組相比,30mmol/L高糖呈時(shí)間依賴的下調(diào)Nephrin以及Podocin的表達(dá),上調(diào)Desmin的表達(dá),且在24h達(dá)高峰。與高糖組相比,1,25(OH)2D3呈濃度依賴的上調(diào)Nephrin以及Podocin的表達(dá),下調(diào)Desmin的表達(dá),且100 nmol/L1,25(OH)2D3的濃度效果最佳。上述實(shí)驗(yàn)結(jié)果證實(shí),足細(xì)胞損傷是糖尿病腎病的早期典型特征,活性維生素D3對高濃度葡萄糖狀態(tài)下足細(xì)胞損傷有保護(hù)作用。 (2)進(jìn)一步探討Nephrin-PI3K/p-Akt信號通路在活性維生素D3保護(hù)足細(xì)胞中的作用發(fā)現(xiàn),與NC組相比,DN組PI3K-p85、 p-Akt表達(dá)量均明顯減低(均P0.05)。與DN組相比,DN+VD組PI3K-p85、p-Akt表達(dá)量均明顯增加(均P0.05),相關(guān)性分析顯示,Nephrin與PI3K-p85及p-Akt呈明顯的正相關(guān)(r=0.736,P0.05;r=0.855,P0.05)。高糖干預(yù)足細(xì)胞24h后,PI3K、p-Akt蛋白表達(dá)明顯減少,給予1,25(OH)2D3可顯著上調(diào)PI3K, p-Akt蛋白的表達(dá),而對總的Akt蛋白的表達(dá)無影響,在1,25(OH)2D3加用PI3K抑制劑LY294002后,足細(xì)胞的損傷尚未得到改善,與高糖組相似。(3)觀察在活性維生素D3治療糖尿病腎病足細(xì)胞損傷的一個(gè)可能的關(guān)鍵靶點(diǎn)TRPC6的表達(dá)變化發(fā)現(xiàn),與NC組相比,DN組VDR表達(dá)量均明顯減低(P0.05),而TRPC6表達(dá)顯著升高。與DN組相比,DN+VD組VDR表達(dá)量明顯增加(P0.05),而TRPC6下調(diào)(P0.05)。相關(guān)性分析顯示,TRPC6與Nephrin(r=-0.752、P0.05)、VDR(r=-0.685、P0.05)呈負(fù)相關(guān),而與24小時(shí)尿蛋白(r=0.739、P0.05)、Desmin (r=0.947、P0.05)呈正相關(guān)。高糖干預(yù)足細(xì)胞24h后,VDR mRNA以及蛋白表達(dá)明顯減少,TRPC6 mRNA以及蛋白表達(dá)明顯增加,給予1,25(OH)2D3可顯著上調(diào)VDR,減少TRPC6表達(dá)。給予TRPC6抑制劑SKF96365干預(yù)后,足細(xì)胞的損傷得到改善,同時(shí)伴隨PI3K、p-Akt蛋白的表達(dá)上調(diào)。結(jié)論1)足細(xì)胞損傷是糖尿病腎病的早期典型病理特征,活性維生素D3對高濃度葡萄糖狀態(tài)下足細(xì)胞損傷有保護(hù)作用。2)高濃度葡萄糖環(huán)境下,Nehrin-PI3K/p-Akt信號通路的下調(diào)介導(dǎo)足細(xì)胞損傷,活性維生素D3能夠通過穩(wěn)定該信號通路減輕足細(xì)胞損傷,從而緩解DN的進(jìn)展。3)高濃度葡萄糖狀態(tài)下TRPO6表達(dá)增高在足細(xì)胞損傷過程中起著重要作用,阻斷THPO6的高表達(dá)可能是活性維生素D3調(diào)節(jié)Nephrin-PI3K-p-Akt信號通路發(fā)揮足細(xì)胞保護(hù)作用的關(guān)鍵環(huán)節(jié)。
[Abstract]:Objective: diabetic nephropathy is a serious complication of diabetes mellitus, which is the main cause of death and disability in patients with diabetic nephropathy. Podocyte injury is considered to be the key factor to cause diabetic nephropathy proteinuria and glomerular sclerosis. Therefore, to explore the mechanism of podocyte injury and drug intervention for injury of podocyte effective is the key to solve the problem. The activity of vitamin D3 has significant protective effect on kidney of diabetic nephropathy, but its mechanism has not been elucidated. This study constructed two culture model induced by STZ model and in vitro in podocytes of diabetic nephropathy rats, to observe the protective effect of vitamin D3 on diabetic nephropathy podocyte, and explore the possible mechanism. Methods: SD male rats rats were randomly divided into four groups: normal control group (group NC), the activity of vitamin D3 intervention group (VD group, 0.1 g ossification in three alcohol kg-1 D-1 gavage), sugar Diabetic nephropathy group (DN group, intraperitoneal injection of STZ 58mg - kg-1) and diabetic nephropathy + vitamin D3 group (group DN+VD), regular blood glucose, body weight, urine samples were collected at the end of the intervention of 18W killed animal, detection of Scr, BUN, Ca, P and 24 hours urinary protein changes, renal pathological changes PAS and MASSON staining, electron microscope observation of podocyte ultrastructure changes. In vitro studies using cultured immortalized mouse podocytes in vitro differentiation of podocytes to different stimuli, observation of 1,25 (OH) 2D3, TRPC6 inhibitor SKF-96365 and PI3K inhibitor effect of LY294002 on high glucose induced podocyte injury. Immunohistochemistry in renal tissue WT-1, Nephrin, Podocin, Desmin, VDR and p-Akt; immunofluorescence detection of the expression of TRPC6 and Nephrin in podocyte, Podocin, Desmin and TRPC6; Real-time PCR Western Blot VDR, renal tissue detection, podocyte protein Nephrin, Podocin, The expression of TRPC6, Desmin and PI3K/p-Akt signal pathway related molecular markers podocytes. Results (1) in vivo, compared with the DN group, DN+VD group, urinary protein decreased up to 36%, reduce the renal pathological damage, reduce blood glucose, podocyte injury, no difference between body mass and blood urea nitrogen (P0.05); serum creatinine calcium, phosphorus, there was no difference between the four groups (all P0.05). Compared with NC group, DN group, Podocin, Nephrin expression was significantly decreased (P0.05), the amount of protein expression of Desmin in podocyte injury markers increased significantly (P0.05). Compared with DN group, DN group, +VD Podocin, Nephrin expression was significantly increase (P0.05), the amount of expression of Desmin in podocyte injury markers were significantly lower (P0.05).WT-1 there was no significant difference between the four groups. In vitro studies confirmed that, compared with the control group, high glucose 30mmol/L in a time dependent downregulation of Nephrin and Podocin expression, upregulating the expression of Desmin, And reached the peak at 24h. Compared with the high glucose group, 1,25 (OH) 2D3 showed a concentration dependent upregulation of Nephrin and Podocin expression, down regulating the expression of Desmin, and 100 nmol/L1,25 (OH) concentration of 2D3 have the best effect. The experimental results showed that podocyte injury is characteristic of early diabetic nephropathy, active vitamin D3 the protective effect of high glucose induced podocyte injury. (2) to further explore the role of Nephrin-PI3K/p-Akt signaling pathway in the activity of vitamin D3 protects podocytes found that compared with the NC group, DN group, PI3K-p85, p-Akt expression was significantly decreased (P0.05). Compared with DN group, DN+VD group, PI3K-p85, p-Akt expression increased significantly (P0.05), correlation analysis showed that Nephrin was significantly correlated with PI3K-p85 and p-Akt (r=0.736, P0.05; r=0.855, P0.05). High glucose foot cells after 24h, PI3K, p-Akt protein expression was significantly reduced to 1,25 (OH 2D3) significantly increased PI3K, p-Akt protein expression, but had no effect on the expression of total Akt protein, 1,25 (OH) PI3K inhibitor LY294002 2D3, podocyte injury has yet to be improved, similar to the high glucose group. (3) found to observe the expression change of a key target of TRPC6 the damage of active vitamin D3 treatment of diabetic kidney disease podocyte, compared with NC group, DN group, VDR expression was significantly decreased (P0.05), and the expression of TRPC6 was significantly increased. Compared with the DN group, DN+VD group, VDR expression was significantly increased (P0.05), and down-regulation of TRPC6 (P0.05). Correlation analysis showed that TRPC6 and Nephrin (r=-0.752, P0.05), VDR (r=-0.685, P0.05) was negatively correlated with 24 hours urine protein (r=0.739, P0.05), Desmin (r=0.947, P0.05) were positively correlated to high glucose. Podocyte 24h, expression of VDR mRNA and protein significantly reduced expression of TRPC6 protein and mRNA increased significantly, given the 1,25 (OH 2D3) significantly increased VDR, reduced the expression of TRPC6. With the TRPC6 inhibitor SKF96365 intervention, podocyte injury improved, accompanied by PI3K, increase the expression of p-Akt. Conclusion: 1) the podocyte injury is the typical pathological features of early diabetic nephropathy, active vitamin D3 has a protective effect of.2 on high glucose under foot cell injury) high glucose environment, Nehrin-PI3K/p-Akt signaling pathway mediated down-regulation of podocyte injury, the activity of vitamin D3 can reduce podocyte injury by stabilizing the signaling pathway, so as to relieve the progress of.3 DN) high glucose increased expression of TRPO6 plays an important role in podocyte injury in the process of blocking high expression of THPO6 the activity of vitamin D3 may be a key step in regulating Nephrin-PI3K-p-Akt signaling pathway play a protective effect of podocytes.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R587.2;R692.9

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