發(fā)布時(shí)間：2018-07-05 00:19

  本文選題：膜性腎�。∕N） + 陽(yáng)離子化牛血清白蛋白（C-BSA）��； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：膜性腎病(membranons nephropathy，MN)是成人性腎病綜合征的常見(jiàn)病因,其病理特點(diǎn)為局限于腎小球上皮下免疫復(fù)合物沉積。臨床表現(xiàn)為大量蛋白尿。本研究通過(guò)注射陽(yáng)離子化牛血清白蛋白的方法，建立SD大鼠膜性腎病模型，并應(yīng)用舒洛地特進(jìn)行干預(yù)，觀察大鼠24小時(shí)尿蛋白定量以及膜性腎病腎小球podocin、CD2AP、nephrin表達(dá)的變化，從而探討舒洛地特對(duì)膜性腎病干預(yù)的新靶點(diǎn)，為人類防治膜性腎病提供新的理論依據(jù)。 方法：選擇8周齡清潔級(jí)健康雄性SD大鼠40只，體重160±20g，普通飼料適應(yīng)性飼養(yǎng)1周后，測(cè)24小時(shí)尿蛋白均為正常，采用隨機(jī)法將其分為4組，正常對(duì)照組、模型組、高劑量舒洛地特組、低劑量舒洛地特組，每組10只。除正常對(duì)照組外，其余組應(yīng)用自備陽(yáng)離子化牛血清白蛋白（C-BSA）預(yù)免疫1周后，隔日尾靜脈注射C-BSA復(fù)制膜性腎病大鼠模型。正常對(duì)照組尾靜脈注射等體積生理鹽水，連續(xù)4周后代謝籠中留取各組大鼠24小時(shí)尿液，檢測(cè)24小時(shí)尿蛋白定量。造模成功后高劑量舒洛地特組給予舒洛地特20mg/kg灌胃，低劑量舒洛地特組給予舒洛地特10mg/kg灌胃。模型組和正常對(duì)照組灌服等體積生理鹽水，各組大鼠自由進(jìn)食、飲水。連續(xù)給藥4周后，再次留取各組大鼠的24小時(shí)尿液，檢測(cè)24小時(shí)尿蛋白定量，隨后將大鼠處死，留取腎組織，應(yīng)用光鏡、電鏡觀察大鼠腎小球病變情況，采用免疫組織化學(xué)方法檢測(cè)podocin、CD2AP、nephrin的表達(dá)情況，結(jié)果應(yīng)用圖像分析系統(tǒng)IPP(Image-Pro Plus)進(jìn)行半定量分析。 結(jié)果： 1.實(shí)驗(yàn)大鼠的一般情況 正常對(duì)照組的大鼠飲食、尿量均正常，反應(yīng)靈敏，眼睛有神，肌肉豐滿。其余三組大鼠經(jīng)尾靜脈注射C-BSA后，出現(xiàn)飲食減少、精神萎靡、毛發(fā)脫落、少動(dòng)抱團(tuán)，大部分大鼠出現(xiàn)陰囊水腫。實(shí)驗(yàn)最后一周時(shí)，與正常對(duì)照組相比，造模各組大鼠體形較小，毛發(fā)脫落（腹部為重）。 2.24小時(shí)尿蛋白定量 24小時(shí)尿蛋白定量，在連續(xù)尾靜脈注射C-BSA4周后，模型組、高劑量舒洛地特組、低劑量舒洛地特組與正常對(duì)照組比較均顯著升高（P均0.05），連續(xù)舒洛地特灌胃4周后，高劑量舒洛地特組與低劑量舒洛地特組24小時(shí)尿蛋白較前均有所降低，與模型組相比有顯著性差異（P均0.05），與低劑量舒洛地特組相比，高劑量舒洛地特組24小時(shí)尿蛋白降低更明顯（P0.05）。 3.腎組織病理形態(tài)學(xué)觀察 光鏡：正常對(duì)照組大鼠腎小球形態(tài)基本正常，毛細(xì)血管袢開(kāi)放良好，無(wú)固有細(xì)胞增多，無(wú)基底膜增厚；模型組大鼠腎小球固有細(xì)胞增多，基底膜明顯增厚；高劑量舒洛地特組和低劑量舒洛地特組大鼠的腎小球固有細(xì)胞輕度增多，基底膜增厚較模型組減輕。 電鏡：正常對(duì)照組大鼠的足細(xì)胞結(jié)構(gòu)清晰，足突排列整齊；模型組大鼠的腎小球基底膜彌漫增厚，上皮下電子致密物沉積，足細(xì)胞足突廣泛融合或消失；低劑量舒洛地特組大鼠腎小球基底膜不規(guī)則增厚，上皮下電子致密物沉積，足突部分融合；高劑量舒洛地特組大鼠基底膜輕度增厚，上皮下少量電子致密物沉積，足突部分融合。 4.免疫組化結(jié)果 從免疫組化染色中可以觀察到podocin、CD2AP、nephrin三種蛋白表達(dá)于腎小球上皮下。該三種蛋白在正常對(duì)照組腎小球上均有大量表達(dá)，模型組、低劑量舒洛地特組及高劑量舒洛地特組腎小球上也均有表達(dá)，但較正常對(duì)照組表達(dá)降低，有統(tǒng)計(jì)學(xué)意義（P均0.05），模型組中podocin、CD2AP、nephrin的表達(dá)較低劑量舒洛地特組及高劑量舒洛地特組表達(dá)均降低，，有統(tǒng)計(jì)學(xué)意義（P均0.05），低劑量舒洛地特組與高劑量舒洛地特組相比，podocin、CD2AP、nephrin表達(dá)較低，具有統(tǒng)計(jì)學(xué)意義（P均0.05）。 結(jié)論： 1.膜性腎病大鼠足細(xì)胞nephrin、podocin及CD2AP表達(dá)下降，并與足細(xì)胞足突融合程度及蛋白尿程度變化一致。提示nephrin、podocin及CD2AP可能參與膜性腎病腎小球足細(xì)胞損傷和蛋白尿產(chǎn)生。 2.舒洛地特可以降低膜性腎病大鼠24小時(shí)尿蛋白含量。 3.舒洛地特可以上調(diào)膜性腎病大鼠腎組織中podocin、CD2AP、nephrin的表達(dá)，提示舒洛地特對(duì)膜性腎病的腎臟保護(hù)作用可能與維持足細(xì)胞裂隙隔膜結(jié)構(gòu)和腎小球?yàn)V過(guò)功能的完整性有關(guān)。 4.舒洛地特治療大鼠膜性腎病尿蛋白及腎臟保護(hù)作用具有劑量依懶性。
[Abstract]:Objective: membranons nephropathy (MN) is a common cause of adult nephrotic syndrome. Its pathological features are limited to the deposition of subcutaneous immuno complex on the glomeruli. The clinical manifestation is a large number of proteinuria. This study established a model of membranous nephropathy in SD rats by injection of cationic bovine serum albumin and applied sulo. To observe the changes of the 24 hour urine protein and the expression of podocin, CD2AP and nephrin in the glomeruli of membranous nephropathy, and to explore the new target for the intervention of membranous nephropathy and provide a new theoretical basis for the prevention and treatment of membranous nephropathy in human.
Methods: 40 healthy male SD rats of 8 weeks old were selected, the body weight was 160 + 20g. After 1 weeks of common feed adaptation, the urine protein was normal in 24 hours. The normal control group was divided into 4 groups, the normal control group, the model group, the high dose sulodiide group and the low dose sulodiide group, with 10 rats in each group. The other groups were used in addition to the normal control group. After 1 weeks of pre immunization with cationic bovine serum albumin (C-BSA), the rat model of membranous nephropathy was replicated by intravenous injection of C-BSA in the tail vein of the next day. The normal control group was injected with the same volume of normal saline in the caudal vein for 4 weeks. The urine of 24 hours of urine was left in each group of offspring, and the urine protein was measured for 24 hours. The high dose of sulalate group was given after the success of the model. The sullock 20mg/kg was given to the stomach. The low dose sullock group was given the sullock 10mg/kg. The model group and the normal control group were given the same volume of normal saline. The rats in each group were free to eat and drinking water. After 4 weeks of continuous administration, the urine of the rats in each group was retained for 24 hours, and the urine protein was measured at 24 hours. Then the rats were killed and the kidney group was left to take the kidney group. The expression of podocin, CD2AP and nephrin was detected by immunohistochemical method, and the results of immunohistochemical staining were used to detect the expression of podocin, CD2AP and nephrin. The results were semi quantitative analysis by using the image analysis system IPP (Image-Pro Plus).
Result:
1. the general situation of experimental rats
The rats in the normal control group have normal diet, the urine volume is normal, the reaction is sensitive, the eyes have the God, the muscle is plump. After the tail vein injection of C-BSA in the rest of the rats, the diet decreases, the spirit is dispirited, the hair falls off, the scrotal edema is seldom moved. The last week of the experiment, compared with the normal control group, the shape of the model rats is compared with the normal control group, the shape of the model rats is compared with the normal control group, the shape of the model rats is compared with the normal control group, the model rats body shape is compared with the normal control group. Small, hair shedding (the abdomen is heavy).
2.24 hour urine protein quantitative
24 hours urine protein quantitative, after C-BSA4 weeks of continuous tail vein injection, model group, high dose suludate group, low dose suludate group and normal control group were significantly higher (P 0.05). After 4 weeks of continuous sulo gavage, the high dose of sullock group and low dosage of sullock group 24 hours urine protein lower than before, and the model. There was a significant difference between the two groups (P = 0.05). Compared with the low dose sullow group, the 24 hour urine protein of the high dose sullow group decreased more significantly (P0.05).
Pathological observation of 3. renal tissues
In the normal control group, the glomerular morphology of the normal control group was basically normal, the capillary loops were open well, no more innate cells and thickening of the basement membrane. The glomerular innate cells in the model group were increased and the basement membrane was thickened, and the glomerular innate cells in the high dose sullad group and low dose sullad group were slightly increased and the basement membrane was slightly increased. The thickening is less than that of the model group.
In the normal control group, the podocyte structure was clear and the foot process was arranged neatly. The glomerular basement membrane in the model group was thickened with thickening of the glomerular basement membrane, the epithelia electron dense deposits were deposited, the podocyte poddate was widely fused or disappeared; the glomerular basement membrane unconventional in the low dose suludate group was thickened, the epithelia electron dense deposit, the foot process In the high dose sulsulide group, the basement membrane was slightly thickened, a small amount of electron dense deposits under the epithelium, and some fusion of foot processes.
4. immunohistochemical results
Three proteins of podocin, CD2AP, and nephrin were expressed in the subcutaneous of glomeruli from immunohistochemical staining. The three proteins were expressed in a large number of glomeruli in the normal control group. In the model group, the glomeruli were also expressed in the low dose sulosate group and the high dose sulosate group, but the expression was lower than that in the normal control group, and the statistics were statistically lower. Statistics showed that there was a statistical difference between the normal control group and the normal control group. The significance (P 0.05), the expression of podocin, CD2AP, nephrin in the model group was lower than the low dose sullock group and high dose sullock group. The expression was statistically significant (P 0.05). The low dose sullock group was lower in podocin, CD2AP, nephrin than in the high dose sullock group (P 0.05).
Conclusion:
The expression of nephrin, podocin and CD2AP in the podocytes of 1. membranous nephrotic rats decreased, and was consistent with the degree of podocyte fusion and proteinuria. It suggested that nephrin, podocin and CD2AP may be involved in the damage of glomerulonephritis and the production of proteinuria in membranous nephropathy.
2. sulpiride could reduce the 24 hour urine protein level in membranous nephropathy rats.
3. sulonate can increase the expression of podocin, CD2AP and nephrin in renal tissue of rats with membranous nephropathy, suggesting that the protective effect of sulonate on membranous nephropathy may be related to the maintenance of the integrity of the structure of the septum septum and the integrity of glomerular filtration function.
4. sulpiride treatment of urinary albumin and renal protection in rats with membranous nephropathy is dose dependent.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R692

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本文編號(hào)：2098096