本文關(guān)鍵詞：雷公藤多苷對糖尿病腎病大鼠腎小管間質(zhì)激活素A表達及轉(zhuǎn)分化的研究機制　出處：《山西醫(yī)科大學》2014年碩士論文　論文類型：學位論文

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【摘要】：目的：通過鏈脲佐菌素（STZ）誘導的糖尿病腎�。―N）實驗大鼠動物模型的建立，觀察高糖刺激下腎臟組織中激活素A(Act-A)的表達水平及腎小管間質(zhì)上皮轉(zhuǎn)分化相關(guān)蛋白α平滑肌肌動蛋白（α-SMA）、E-鈣粘蛋白(E-cad）和腎臟纖維化的相關(guān)蛋白Ⅳ型膠原（Co-IV）的表達情況，用雷公藤多苷片對DN大鼠進行干預實驗后，觀察上述蛋白表達的變化情況，進一步探討糖尿病腎病腎小管間質(zhì)上皮轉(zhuǎn)分化及腎臟纖維化的發(fā)病機制，以及雷公藤多苷片對這一病理過程的保護作用機制。 方法：（1）選取清潔級Wistar雄性大鼠40只，體重180-200g，6周齡，隨機分為正常對照組(A組)、糖尿病腎病模型組(B組)、厄貝沙坦治療組（C組）、雷公藤多苷治療組（D組），每組實驗大鼠10只，正常對照組的實驗大鼠給予腹腔注射等劑量的檸檬酸緩沖液，其余各組實驗大鼠給予腹腔注射鏈脲佐菌素（STZ，55mg/kg），誘導成糖尿病模型，腹腔注射72小時后，尾靜脈采血，以連續(xù)三次隨機血糖（BG）≥16.7mmol/L,尿糖≥4+作為糖尿病大鼠模型建立成功的標準。糖尿病模型造模成功四周后檢測24小時尿蛋白定量，測定24小時尿蛋白大于30mg時即認為糖尿病腎病的模型建模成功。 （2）各組實驗大鼠成模后，雷公藤多苷治療組的實驗大鼠給予雷公藤多苷混懸液0.5mg/100g.d灌胃治療，厄貝沙坦陽性對照組的實驗大鼠給予厄貝沙坦混懸液17.5mg/kg.d灌胃治療，正常對照組和糖尿病腎病模型組的實驗大鼠給予等量蒸餾水灌胃。 （3）實驗第4、8、12、16周末秤取大鼠體質(zhì)量、尾靜脈采血測定血糖，將代謝籠收集好的24小時尿液檢測24h尿蛋白(24hPro)定量，第16周末腹腔麻醉處死大鼠，開胸后心尖部抽血，分離血清，檢測血肌酐(Scr)和血糖，，切取腎臟組織并稱重，計算腎臟肥大指數(shù)；取大鼠腎臟組織行HE、PAS染色，進行病理組織學觀察。 （4）用免疫組化法檢測腎小管間質(zhì)中激活素A（Act-A）、α平滑肌肌動蛋白(α-SMA）、Ⅳ型膠原（Co-IV）及E-鈣粘蛋白(E-cad）的表達。 （5）采用實時熒光定量PCR技術(shù)檢測腎小管間質(zhì)中激活素A（Act-A）、α平滑肌肌動蛋白(α-SMA）、Ⅳ型膠原（Co-IV）及E-鈣粘蛋白(E-cad）蛋白mRNA的表達。 結(jié)果：（1）16周后厄貝沙坦治療組及雷公藤多苷治療組的實驗大鼠24h尿蛋白、血肌酐（Scr）和腎臟肥大指數(shù)均低于糖尿病腎病模型組(P均＜0.05)，其中厄貝沙坦治療組和雷公藤多苷治療組之間比較，差異無統(tǒng)計學意義。 （2）正常對照組的實驗大鼠腎小管形態(tài)、結(jié)構(gòu)、分布以及腎小球結(jié)構(gòu)，均未出現(xiàn)明顯的病理改變。與糖尿病腎病模型組比較，厄貝沙坦治療組和雷公藤多苷治療組的實驗大鼠體質(zhì)量增加，病理切片均顯示腎小管間質(zhì)病變程度減輕，厄貝沙坦治療組和雷公藤多苷治療組之間比較，差異無統(tǒng)計學意義。 （3）與糖尿病腎病模型組比較，免疫組化和實時熒光定量PCR均顯示，厄貝沙坦治療組和雷公藤多苷治療組腎小管間質(zhì)激活素A（Act-A）、α平滑肌肌動蛋白(α-SMA）和Ⅳ型膠原（Co-IV）的表達明顯下降(P＜0.01)，E-鈣粘蛋白(E-cad）的表達明顯上升(P＜0.01)，但厄貝沙坦治療組和雷公藤多苷治療組之間比較，差異無統(tǒng)計學意義。 結(jié)論：雷公藤多苷（TWP）可以降低糖尿病腎病24小時尿蛋白的排泄，改善腎小管間質(zhì)的損傷，具有一定的腎臟保護作用，激活素A（Act-A）在糖尿病腎病中具有誘導腎小管間質(zhì)上皮細胞轉(zhuǎn)分化的作用，同時能促進腎臟發(fā)生纖維化，通過下調(diào)激活素A（Act-A）的表達，從而阻礙腎小管間質(zhì)上皮細胞的轉(zhuǎn)分化，延緩腎臟發(fā)生纖維化的進程。
[Abstract]:Objective: by streptozotocin (STZ) induced diabetic nephropathy (DN) to establish the experimental rat model of animal, were stimulated by high glucose in the kidney tissues of activin A (Act-A) and the expression level of renal tubular interstitial epithelial transdifferentiation of alpha smooth muscle actin related protein (-SMA alpha), E- cadherin (E-cad) and related proteins in renal fibrosis collagen type IV (Co-IV) expression, intervention experiment of DN rats with Tripterygium wilfordii tablets, observed the protein expression changes, further study of diabetic renal interstitial epithelial transdifferentiation and renal fibrosis in the pathogenesis and mechanism of Tripterygium the protective effect of the pathological process.
Methods: (1) select clean level 40 male Wistar rats, weight 180-200g, 6 weeks old, were randomly divided into normal control group (A group), diabetic nephropathy model group (group B), irbesartan treatment group (group C), Tripterygium wilfordii group (D group), each group of rats 10 rats, citric acid buffer in rats of normal control group received intraperitoneal dose, the rest rats were given intraperitoneal injection of streptozotocin (STZ, 55mg/kg), diabetic model induced by intraperitoneal injection, after 72 hours, the tailvein, with three consecutive random blood glucose (BG) is more than 16.7mmol/L, more than 4+ of urine as a diabetic rat model was established successfully. The standard of quantitative measurement of 24 hours urinary protein in diabetic model rats after four weeks, the determination of 24 hours urine protein greater than 30mg that the model of diabetic nephropathy.
(2) in experimental rats after modeling, rats GTW treatment group given Tripterygium wilfordii 0.5mg/100g.d gavage treatment, the rats of irbesartan in the positive control group given irbesartan 17.5mg/kg.d gavage treatment, the normal control group and diabetic nephropathy model group rats given the equal volume of distilled water.
(3) the 4,8,12,16 weekend weighed the body mass of rats, blood glucose of tail vein was 24 hours urine protein and 24h urine will collect good metabolic cages (24hPro) quantitative, sixteenth weeks were anesthetized rats were sacrificed after thoracotomy, apex blood, separation of serum, blood creatinine (Scr) and blood glucose, cut kidney tissue and weighed to calculate the renal hypertrophy index; rats kidney tissue HE, PAS staining, and histopathology.
(4) detected tubulointerstitial activin A immune group (Act-A), alpha smooth muscle actin (alpha -SMA), collagen type IV (Co-IV) and E- cadherin (E-cad) expression.
(5) using real-time fluorescence quantitative PCR detection of renal tubulointerstitial activin A (Act-A), alpha smooth muscle actin (alpha -SMA), collagen type IV (Co-IV) and E- cadherin (E-cad) expression of mRNA protein.
Results: (1) 16 weeks after irbesartan treatment group and Tripterygium wilfordii group rats, 24h urine protein, serum creatinine (Scr) and renal hypertrophy index were lower than those in diabetic nephropathy model group (P < 0.05), which between irbesartan treatment group and tripterygium glycosides group, no difference statistical significance.
(2) normal control group rat renal tubular morphology, structure, distribution and glomeruli, there were no significant pathological changes. Compared with diabetic nephropathy model group, irbesartan treatment group and tripterygium glycosides more body mass treatment group rats increased, pathological slices showed the degree of tubulointerstitial lesions were alleviated and the comparison between irbesartan group and twp treatment group, the difference was not statistically significant.
(3) compared with diabetic nephropathy model group, immunohistochemistry and real-time fluorescence quantitative PCR showed that irbesartan treatment group and Tripterygium wilfordii group tubulointerstitial activin A (Act-A), alpha smooth muscle actin (alpha -SMA) and collagen type IV (Co-IV) expression significantly decreased (P < 0.01) E- (E-cad), E-cadherin expression was significantly increased (P < 0.01), but between irbesartan treatment group and twp treatment group, the difference was not statistically significant.
Conclusion: tripterygium glycosides (TWP) can reduce diabetic nephropathy 24 hours urinary protein excretion, improve renal tubulointerstitial injury has some protective effect of kidney, activin A (Act-A) is induced by interstitial Epithelial Cells Transdifferentiation for renal tubule in diabetic nephropathy, and can promote kidney fibrosis. Through down-regulation of activin A (Act-A) expression, thus impeding the transdifferentiation of renal tubular epithelial cell mass, retard renal fibrosis process.

【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R587.2;R692

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