本文選題：糖尿病腎病 + 叉頭狀轉(zhuǎn)錄因子O1�。� 參考：《鄭州大學(xué)》2014年碩士論文

【摘要】：背景與目的 糖尿病腎病(DN)是糖尿病(DM)微血管并發(fā)癥之一，常導(dǎo)致終末期腎病。DN足細(xì)胞損傷常表現(xiàn)為足突融合消失、細(xì)胞體積逐漸變小、陰離子電荷減少，最終足細(xì)胞可從腎小球基底膜(glomerular basement membrane,GBM)上脫落到腎小囊里并從尿液中排出[1]。目前，，隨著足細(xì)胞系的建立，足細(xì)胞損傷在DN發(fā)病中被認(rèn)為是引起DN蛋白尿和腎小球硬化的關(guān)鍵[2]。 叉頭狀轉(zhuǎn)錄因子O1（Forkhead transcription factor O1，F(xiàn)oxO1）是FoxO家族中的一員，當(dāng)被磷酸化后可導(dǎo)致其轉(zhuǎn)錄活性降低，引起FoxO1靶基因的表達(dá)下降[3]。本課題組前期實(shí)驗(yàn)[4]發(fā)現(xiàn)DN大鼠腎皮質(zhì)FoxO1表達(dá)及活性下降，隨后[5]又發(fā)現(xiàn)DN治療組大鼠腎臟的FoxO1較未治療組磷酸化水平顯著降低，活性顯著增強(qiáng)，電鏡下足突融合減輕，提示FoxO1的活性變化可能和DN中足細(xì)胞損傷情況有關(guān)，但并沒(méi)有驗(yàn)證兩者的直接關(guān)系，目前國(guó)內(nèi)外也沒(méi)有相關(guān)報(bào)道。另有文獻(xiàn)[6]報(bào)道，PI3K/Akt信號(hào)通路可能參與了DN足細(xì)胞損傷的發(fā)生與發(fā)展。Foxo1作為PI3K/Akt信號(hào)通路的下游因子，我們認(rèn)為其活性的改變很可能也參與了DN足細(xì)胞的損傷過(guò)程。 本研究旨在通過(guò)上調(diào)FoxO1的表達(dá)，觀察FoxO1對(duì)糖尿病大鼠足細(xì)胞的影響。 材料與方法 健康、雄性清潔級(jí)SD大鼠120只，體重(100±20)g，IVC系統(tǒng)中喂養(yǎng)。待大鼠達(dá)到8周齡，體重（220±20）g，隨機(jī)選取90只建立DM大鼠模型，并隨機(jī)分為DM+空慢病毒(LV-pSC-GFP)感染組（a組，n=30），DM+大鼠結(jié)構(gòu)性活性FoxO1慢病毒（LV-CA-FoxO1）感染組（b組，n=30），DM組（d組，n=30）。剩余大鼠注射相等體積的檸檬酸鈉-檸檬酸緩沖液作為正常對(duì)照組（c組，n=30）。DM大鼠造模方法如下：將其禁食12h后，按60mg/kg一次性腹腔注射1%STZ溶液，72h后連續(xù)三次測(cè)定血糖（BG）≥16.7mmol/L，為DM成模標(biāo)準(zhǔn)。各組大鼠自由進(jìn)食飲水，不給予任何降糖藥物治療。待DM大鼠造模成功、血糖穩(wěn)定5天后，采用腎臟多部位靶向注射重組慢病毒的方法[7]分別將100ulLV-pSC-GFP、LV-CA-FoxO1（慢病毒滴度檢測(cè)為7×108TU/ml，綜合考慮慢病毒感染效率及細(xì)胞毒性后明確感染大鼠的最佳感染量為100ul）注射到a、b組大鼠的右腎皮質(zhì)多個(gè)部位。c組和d組注射等量的生理鹽水。于感染后的2、4、8w末，將各組大鼠置于代謝籠收集24小時(shí)尿，離心后保存于4℃冰箱，用來(lái)測(cè)定24小時(shí)尿蛋白（UPro/24h）、尿白蛋白定量（UAlb）以及尿沉渣中足盂蛋白（podocalyxin, PCX）的含量。麻醉后內(nèi)眥靜脈取血，分離血清檢測(cè)血肌酐（Scr）、尿素氮（BUN）。處死大鼠取出右腎去包膜測(cè)腎重并進(jìn)行以下取材。a、b兩組大鼠取右腎背側(cè)下1/2做冰凍切片以及光鏡切片，觀察慢病毒感染情況，c、d組直接將切下的背側(cè)下1/2組織置于4%多聚甲醛中固定用于制作光鏡切片觀察腎小球病理學(xué)變化。a、b、c、d四組取米粒(約1mm3)大小腎組織（包括注射點(diǎn)在內(nèi)）迅速放入冷戊二醛固定液中用于電鏡觀察腎臟超微結(jié)構(gòu)變化，剩余腎組織迅速置于-80℃冰箱里，用于Real-time PCR和Western blotting檢測(cè)FoxO1、足盂蛋白（podocalyxin，PCX）、nephrin、Ⅳ型膠原α3鏈（COL4A3）、Ⅳ型膠原α5鏈（COL4A5）、結(jié)蛋白（desmin）的表達(dá)。 結(jié)果 1.熒光顯微鏡下，可觀察到a組和b組大鼠腎臟冰凍切片中都有綠色熒光蛋白（GFP）的有效表達(dá)，提示慢病毒感染成功。 2.2周末，四組大鼠的UPro/24h、UAlb、Scr、BUN差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）；與c組比較，a、d組BG升高，KI升高（均P0.05），BW降低（P0.05），b組與a、d組比，BG、BW無(wú)明顯差別（P0.05），KI降低（P0.05），但仍高于c組（P0.05）。在4、8周末，與c組相比， a、d組BG、KI、UPro/24h、UAlb、Scr、BUN水平顯著升高，BW水平明顯降低（均P0.05）；與a、d組比較，b組KI、UPro/24h、UAlb、Scr和BUN水平明顯降低（均P0.05），但仍高于c組（P0.05），b組BG水平較a、d組略有下降、BW水平略有上升，但差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。a組和d組各觀察點(diǎn)各項(xiàng)指標(biāo)均無(wú)明顯差異（P0.05）。 3. ELISA結(jié)果：2、4、8周末，與c組相比，a組和d組大鼠尿沉渣中PCX含量顯著增高（P0.05）；與a、d組比較，b組尿沉渣PCX含量明顯下降（P0.05），但仍高于c組（P0.05）；a與d組差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。除c組外，各組該指標(biāo)呈時(shí)間依賴性變化。 4.在2、4、8周末各個(gè)觀察點(diǎn)上，a和d組大鼠腎皮質(zhì)中的FoxO1、nephrin、PCX mRNA表達(dá)水平顯著低于c組（P0.05）；b組大鼠三者mRNA表達(dá)水平顯著高于a、d組（P0.05），但nephrin、PCX的mRNA水平仍低于c組（P0.05），同時(shí)FoxO1顯著高于c組（P0.05）。a組和d組腎皮質(zhì)中COL4A3、COL4A5以及desmin mRNA表達(dá)水平顯著高于c組（P0.05）；b組這三項(xiàng)指標(biāo)較a和d組顯著下降（P0.05），但仍高于c組（P0.05）。a組和d組各項(xiàng)指標(biāo)差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。 5.Western blot結(jié)果顯示，在第2、4、8周末各個(gè)觀察點(diǎn)上，a組和d組較c組大鼠腎皮質(zhì)中的FoxO1蛋白表達(dá)水平無(wú)顯著差異（P0.05），但FoxO1/p-FoxO1、nephrin、 PCX蛋白表達(dá)水平顯著降低（P0.05），b組大鼠腎臟FoxO1、nephrin、PCX蛋白表達(dá)水平顯著高于a、d兩組（P0.05），但nephrin、PCX仍低于c組（P0.05），同時(shí)b組的FoxO1蛋白表達(dá)水平與FoxO1/p-FoxO1顯著高于c組（P0.05）。a、d兩組的COL4A3、COL4A5以及desmin的蛋白表達(dá)水平顯著高于c組（P0.05）；b組的COL4A3、COL4A5以及desmin蛋白這三項(xiàng)指標(biāo)較a和d組顯著下降（P0.05），但仍高于c組（P0.05）。a組和d組各項(xiàng)指標(biāo)差異無(wú)統(tǒng)計(jì)學(xué)意義（P0.05）。 6.腎臟病理變化：光鏡下c組大鼠腎小球結(jié)構(gòu)未見明顯異常，a、d組差異不大，可見腎小球體積增大，系膜細(xì)胞增生，細(xì)胞外基質(zhì)增多，基底膜增厚，以8周末變化最明顯；電鏡下c組大鼠腎小球結(jié)構(gòu)未見明顯異常，未見基底膜及內(nèi)皮細(xì)胞增厚，足突均勻分布，a、d組差異不大，可見腎小球基底膜增厚，厚薄不均勻，足細(xì)胞足突增寬甚至融合消失，8周末變化最為顯著，b組大鼠腎小球病理變化改善明顯。 結(jié)論 上調(diào)FoxO1的表達(dá)可以減輕糖尿病大鼠足細(xì)胞的損傷，其可能是通過(guò)改變足細(xì)胞相關(guān)蛋白（nephrin、PCX、desmin）的表達(dá)實(shí)現(xiàn)的。
[Abstract]:Background and purpose
Diabetic nephropathy (DN) is one of the microvascular complications of diabetes (DM), which often leads to.DN podocyte injury in end-stage renal disease, which often appears as the disappearance of foot process fusion. The cell volume gradually decreases and the anion charge decreases. Finally, the podocyte can fall from the glomerular basement membrane (glomerular basement membrane, GBM) into the renal pouch and discharge from the urine. 1]. now, with the establishment of podocyte, podocyte injury is considered to be the key [2]. causing DN proteinuria and glomerulosclerosis in the pathogenesis of DN.
The fork head transcription factor O1 (Forkhead transcription factor O1, FoxO1) is a member of the FoxO family. When phosphorylated, the transcriptional activity can be reduced and the expression of the target gene of the FoxO1 is reduced and the expression of the target gene of the FoxO1 is reduced. The level of phosphorylation in oxO1 was significantly lower than that in the untreated group. The activity was significantly enhanced and the fusion of foot process was reduced under the electron microscope. It suggested that the changes in the activity of FoxO1 may be related to the condition of the foot cell damage in DN, but there is no direct relationship between the two. And there are no relevant reports at home and abroad. And [6] reports that the PI3K/Akt signaling pathway may be involved in DN The occurrence and development of.Foxo1 as a downstream factor of PI3K/Akt signaling pathway, we believe that the changes in its activity may also be involved in the damage process of DN podblast.
The aim of this study was to observe the effect of FoxO1 on podocytes in diabetic rats by up regulating the expression of FoxO1.
Materials and methods
Healthy, 120 male clean SD rats, weight (100 + 20) g, IVC system, 8 weeks of age and weight (220 + 20) g in rats, 90 rats were randomly selected and randomly divided into DM+ (LV-pSC-GFP) infection group (a group, n=30), FoxO1 lentivirus (LV-CA-FoxO1) infection group of DM+ rats 30). The remaining rats were injected with the equal volume of sodium citrate buffer solution as the normal control group (Group C, n=30).DM rats as follows: after fasting 12h, 1%STZ solution was injected into the abdominal cavity by 60mg/kg and 72h after 72h, and BG (BG) was more than 16.7mmol/L, which was a standard for DM. The rats were free to eat drinking water and were not given. Treatment of any hypoglycemic drugs. After a successful model of DM rats, after 5 days of blood glucose stability, the 100ulLV-pSC-GFP, LV-CA-FoxO1 (lentivirus titer, 7 x 108TU/ml), LV-CA-FoxO1 (lentivirus titer (lentivirus titer), and the optimal infection rate of the infected rats were considered 100ul). The rats in group B were injected with the same amount of normal saline injection in group.C and D of the right renal cortex in group a. At the end of 2,4,8w after infection, the rats were placed in the metabolic cage for 24 hours urine, and then stored at 4 centigrade refrigerators to determine the 24 hour urine protein (UPro/24h), the whiteness of the white egg (UAlb) and the proteinuria in the urine sediment (podocalyxin, P). The content of CX) after anaesthesia, the blood of the canthus vein was taken, serum creatinine (Scr) and urea nitrogen (BUN) were detected by separation serum. The rats were sacrificed to take out the right kidney to measure the kidney weight and take the following material for.A. The B two rats were taken the frozen section of the right renal dorsal 1/2 and the light microscope section to observe the infection of the slow disease. The C, D group directly placed the lower 1/2 tissue under the dorsal side of the incision. 4% polyoxymethylene was used to make light microscopy to observe the pathological changes of glomeruli,.A, B, C, D four groups (about 1mm3) of kidney tissue (including injection points) were quickly put into the cold glutaraldehyde fixation solution to observe the ultrastructural changes of the kidney. The remnant kidney group was quickly placed in the refrigerator of -80 C and used for Real-time PCR and Western. Blotting detected FoxO1, podocalyxin (PCX), nephrin, type IV collagen alpha 3 chain (COL4A3), type IV collagen alpha 5 chain (COL4A5), and the expression of desmin (desmin).
Result
1. under fluorescence microscope, the expression of green fluorescent protein (GFP) in frozen sections of rats in group A and B could be observed, indicating that lentivirus infection was successful.
There was no significant difference in UPro/24h, UAlb, Scr, BUN in the 2.2 weekend rats (P0.05). Compared with the C group, a, D group BG increased, KI increased (P0.05) and BW decreased. As compared with a and D, the level of KI, UPro/24h, UAlb, BUN, Scr and BUN decreased significantly (P0.05) compared with a and D, but it was still higher than that of the C group.
3. ELISA results: on the weekend of 2,4,8, compared with group C, PCX content in a and D group was significantly higher (P0.05). Compared with a and D group, the PCX content of urine sediment in B group decreased significantly (P0.05), but it was still higher than that of the group.
4. at every observation point at 2,4,8 weekend, the expression level of FoxO1, nephrin, PCX mRNA in the renal cortex of a and D rats was significantly lower than that in C group (P0.05), and the mRNA expression level in the three rats of the B group was significantly higher than that of the a group. The expression level of A5 and desmin mRNA was significantly higher than that in group C (P0.05), and the three indexes in group B were significantly lower than those in a and D group (P0.05), but it was still higher than that of C group (P0.05).
The results of 5.Western blot showed that there was no significant difference in the expression level of FoxO1 protein in the renal cortex of the group A and the D group at the observation points at the end of the 2,4,8 weekend (P0.05), but the expression level of FoxO1/p-FoxO1, nephrin, PCX protein decreased significantly (P0.05), and the expression level of the kidneys of the rats was significantly higher than that in the C group. Ephrin, PCX was still lower than group C (P0.05), and the FoxO1 protein expression level and FoxO1/p-FoxO1 in B group were significantly higher than that of C group (P0.05).A. There was no significant difference in indexes between group A and group D (P0.05).
6. renal pathological changes: no obvious abnormal glomerular structure in group C rats was found under light microscope. There was no significant difference in a and D group. The glomerular volume increased, mesangial cells proliferated, the extracellular matrix increased, the basement membrane thickened, and the most obvious changes were in the 8 weekend. Under electron microscope, there was no obvious abnormal glomerular structure in group C and no thickening of basement membrane and endothelial cells. There was no significant difference in the A and D groups. The thickening of the glomerular basement membrane, the uneven thickness of the glomeruli, the widening of the podocyte foot process and even the fusion disappeared, the most significant change in the 8 weekend was observed. The pathological changes in the glomeruli of group B rats were obviously improved.
conclusion
The expression of FoxO1 can reduce the injury of foot cells in diabetic rats, which may be achieved by changing the expression of nephrin (PCX, desmin).
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R587.2

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