FoxO1对糖尿病大鼠足细胞影响的实验研究

发布时间：2018-06-19 00:05

本文选题：糖尿病肾病 + 叉头状转录因子O1　；参考：《郑州大学》2014年硕士论文

【摘要】：背景与目的糖尿病肾病(DN)是糖尿病(DM)微血管并发症之一，常导致终末期肾病。DN足细胞损伤常表现为足突融合消失、细胞体积逐渐变小、阴离子电荷减少，最终足细胞可从肾小球基底膜(glomerular basement membrane,GBM)上脱落到肾小囊里并从尿液中排出[1]。目前，，随着足细胞系的建立，足细胞损伤在DN发病中被认为是引起DN蛋白尿和肾小球硬化的关键[2]。叉头状转录因子O1（Forkhead transcription factor O1，FoxO1）是FoxO家族中的一员，当被磷酸化后可导致其转录活性降低，引起FoxO1靶基因的表达下降[3]。本课题组前期实验[4]发现DN大鼠肾皮质FoxO1表达及活性下降，随后[5]又发现DN治疗组大鼠肾脏的FoxO1较未治疗组磷酸化水平显著降低，活性显著增强，电镜下足突融合减轻，提示FoxO1的活性变化可能和DN中足细胞损伤情况有关，但并没有验证两者的直接关系，目前国内外也没有相关报道。另有文献[6]报道，PI3K/Akt信号通路可能参与了DN足细胞损伤的发生与发展。Foxo1作为PI3K/Akt信号通路的下游因子，我们认为其活性的改变很可能也参与了DN足细胞的损伤过程。本研究旨在通过上调FoxO1的表达，观察FoxO1对糖尿病大鼠足细胞的影响。材料与方法健康、雄性清洁级SD大鼠120只，体重(100±20)g，IVC系统中喂养。待大鼠达到8周龄，体重（220±20）g，随机选取90只建立DM大鼠模型，并随机分为DM+空慢病毒(LV-pSC-GFP)感染组（a组，n=30），DM+大鼠结构性活性FoxO1慢病毒（LV-CA-FoxO1）感染组（b组，n=30），DM组（d组，n=30）。剩余大鼠注射相等体积的柠檬酸钠-柠檬酸缓冲液作为正常对照组（c组，n=30）。DM大鼠造模方法如下：将其禁食12h后，按60mg/kg一次性腹腔注射1%STZ溶液，72h后连续三次测定血糖（BG）≥16.7mmol/L，为DM成模标准。各组大鼠自由进食饮水，不给予任何降糖药物治疗。待DM大鼠造模成功、血糖稳定5天后，采用肾脏多部位靶向注射重组慢病毒的方法[7]分别将100ulLV-pSC-GFP、LV-CA-FoxO1（慢病毒滴度检测为7×108TU/ml，综合考虑慢病毒感染效率及细胞毒性后明确感染大鼠的最佳感染量为100ul）注射到a、b组大鼠的右肾皮质多个部位。c组和d组注射等量的生理盐水。于感染后的2、4、8w末，将各组大鼠置于代谢笼收集24小时尿，离心后保存于4℃冰箱，用来测定24小时尿蛋白（UPro/24h）、尿白蛋白定量（UAlb）以及尿沉渣中足盂蛋白（podocalyxin, PCX）的含量。麻醉后内眦静脉取血，分离血清检测血肌酐（Scr）、尿素氮（BUN）。处死大鼠取出右肾去包膜测肾重并进行以下取材。a、b两组大鼠取右肾背侧下1/2做冰冻切片以及光镜切片，观察慢病毒感染情况，c、d组直接将切下的背侧下1/2组织置于4%多聚甲醛中固定用于制作光镜切片观察肾小球病理学变化。a、b、c、d四组取米粒(约1mm3)大小肾组织（包括注射点在内）迅速放入冷戊二醛固定液中用于电镜观察肾脏超微结构变化，剩余肾组织迅速置于-80℃冰箱里，用于Real-time PCR和Western blotting检测FoxO1、足盂蛋白（podocalyxin，PCX）、nephrin、Ⅳ型胶原α3链（COL4A3）、Ⅳ型胶原α5链（COL4A5）、结蛋白（desmin）的表达。结果 1.荧光显微镜下，可观察到a组和b组大鼠肾脏冰冻切片中都有绿色荧光蛋白（GFP）的有效表达，提示慢病毒感染成功。 2.2周末，四组大鼠的UPro/24h、UAlb、Scr、BUN差异无统计学意义（P0.05）；与c组比较，a、d组BG升高，KI升高（均P0.05），BW降低（P0.05），b组与a、d组比，BG、BW无明显差别（P0.05），KI降低（P0.05），但仍高于c组（P0.05）。在4、8周末，与c组相比， a、d组BG、KI、UPro/24h、UAlb、Scr、BUN水平显著升高，BW水平明显降低（均P0.05）；与a、d组比较，b组KI、UPro/24h、UAlb、Scr和BUN水平明显降低（均P0.05），但仍高于c组（P0.05），b组BG水平较a、d组略有下降、BW水平略有上升，但差异无统计学意义（P0.05）。a组和d组各观察点各项指标均无明显差异（P0.05）。 3. ELISA结果：2、4、8周末，与c组相比，a组和d组大鼠尿沉渣中PCX含量显著增高（P0.05）；与a、d组比较，b组尿沉渣PCX含量明显下降（P0.05），但仍高于c组（P0.05）；a与d组差异无统计学意义（P0.05）。除c组外，各组该指标呈时间依赖性变化。 4.在2、4、8周末各个观察点上，a和d组大鼠肾皮质中的FoxO1、nephrin、PCX mRNA表达水平显著低于c组（P0.05）；b组大鼠三者mRNA表达水平显著高于a、d组（P0.05），但nephrin、PCX的mRNA水平仍低于c组（P0.05），同时FoxO1显著高于c组（P0.05）。a组和d组肾皮质中COL4A3、COL4A5以及desmin mRNA表达水平显著高于c组（P0.05）；b组这三项指标较a和d组显著下降（P0.05），但仍高于c组（P0.05）。a组和d组各项指标差异无统计学意义（P0.05）。 5.Western blot结果显示，在第2、4、8周末各个观察点上，a组和d组较c组大鼠肾皮质中的FoxO1蛋白表达水平无显著差异（P0.05），但FoxO1/p-FoxO1、nephrin、 PCX蛋白表达水平显著降低（P0.05），b组大鼠肾脏FoxO1、nephrin、PCX蛋白表达水平显著高于a、d两组（P0.05），但nephrin、PCX仍低于c组（P0.05），同时b组的FoxO1蛋白表达水平与FoxO1/p-FoxO1显著高于c组（P0.05）。a、d两组的COL4A3、COL4A5以及desmin的蛋白表达水平显著高于c组（P0.05）；b组的COL4A3、COL4A5以及desmin蛋白这三项指标较a和d组显著下降（P0.05），但仍高于c组（P0.05）。a组和d组各项指标差异无统计学意义（P0.05）。 6.肾脏病理变化：光镜下c组大鼠肾小球结构未见明显异常，a、d组差异不大，可见肾小球体积增大，系膜细胞增生，细胞外基质增多，基底膜增厚，以8周末变化最明显；电镜下c组大鼠肾小球结构未见明显异常，未见基底膜及内皮细胞增厚，足突均匀分布，a、d组差异不大，可见肾小球基底膜增厚，厚薄不均匀，足细胞足突增宽甚至融合消失，8周末变化最为显著，b组大鼠肾小球病理变化改善明显。结论上调FoxO1的表达可以减轻糖尿病大鼠足细胞的损伤，其可能是通过改变足细胞相关蛋白（nephrin、PCX、desmin）的表达实现的。
[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).
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2

【参考文献】