本文關(guān)鍵詞：雷公藤多苷對(duì)糖尿病腎病大鼠腎小管間質(zhì)激活素A表達(dá)及轉(zhuǎn)分化的研究機(jī)制　出處：《山西醫(yī)科大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 雷公藤多苷 糖尿病腎病 激活素A 轉(zhuǎn)分化

【摘要】：目的：通過(guò)鏈脲佐菌素（STZ）誘導(dǎo)的糖尿病腎病（DN）實(shí)驗(yàn)大鼠動(dòng)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(dá)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(dòng)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(dá)情況，用雷公藤多苷片對(duì)DN大鼠進(jìn)行干預(yù)實(shí)驗(yàn)后，觀察上述蛋白表達(dá)的變化情況，進(jìn)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(jī)制，以及雷公藤多苷片對(duì)這一病理過(guò)程的保護(hù)作用機(jī)制。 方法：（1）選取清潔級(jí)Wistar雄性大鼠40只，體重180-200g，6周齡，隨機(jī)分為正常對(duì)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(shí)驗(yàn)大鼠10只，正常對(duì)照組的實(shí)驗(yàn)大鼠給予腹腔注射等劑量的檸檬酸緩沖液，其余各組實(shí)驗(yàn)大鼠給予腹腔注射鏈脲佐菌素（STZ，55mg/kg），誘導(dǎo)成糖尿病模型，腹腔注射72小時(shí)后，尾靜脈采血，以連續(xù)三次隨機(jī)血糖（BG）≥16.7mmol/L,尿糖≥4+作為糖尿病大鼠模型建立成功的標(biāo)準(zhǔn)。糖尿病模型造模成功四周后檢測(cè)24小時(shí)尿蛋白定量，測(cè)定24小時(shí)尿蛋白大于30mg時(shí)即認(rèn)為糖尿病腎病的模型建模成功。 （2）各組實(shí)驗(yàn)大鼠成模后，雷公藤多苷治療組的實(shí)驗(yàn)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(yáng)性對(duì)照組的實(shí)驗(yàn)大鼠給予厄貝沙坦混懸液17.5mg/kg.d灌胃治療，正常對(duì)照組和糖尿病腎病模型組的實(shí)驗(yàn)大鼠給予等量蒸餾水灌胃。 （3）實(shí)驗(yàn)第4、8、12、16周末秤取大鼠體質(zhì)量、尾靜脈采血測(cè)定血糖，將代謝籠收集好的24小時(shí)尿液檢測(cè)24h尿蛋白(24hPro)定量，第16周末腹腔麻醉處死大鼠，開(kāi)胸后心尖部抽血，分離血清，檢測(cè)血肌酐(Scr)和血糖，，切取腎臟組織并稱重，計(jì)算腎臟肥大指數(shù)；取大鼠腎臟組織行HE、PAS染色，進(jìn)行病理組織學(xué)觀察。 （4）用免疫組化法檢測(cè)腎小管間質(zhì)中激活素A（Act-A）、α平滑肌肌動(dòng)蛋白(α-SMA）、Ⅳ型膠原（Co-IV）及E-鈣粘蛋白(E-cad）的表達(dá)。 （5）采用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)腎小管間質(zhì)中激活素A（Act-A）、α平滑肌肌動(dòng)蛋白(α-SMA）、Ⅳ型膠原（Co-IV）及E-鈣粘蛋白(E-cad）蛋白mRNA的表達(dá)。 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(shí)驗(yàn)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(wú)統(tǒng)計(jì)學(xué)意義。 （2）正常對(duì)照組的實(shí)驗(yàn)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(shí)驗(yàn)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(wú)統(tǒng)計(jì)學(xué)意義。 （3）與糖尿病腎病模型組比較，免疫組化和實(shí)時(shí)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(dòng)蛋白(α-SMA）和Ⅳ型膠原（Co-IV）的表達(dá)明顯下降(P＜0.01)，E-鈣粘蛋白(E-cad）的表達(dá)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(wú)統(tǒng)計(jì)學(xué)意義。 結(jié)論：雷公藤多苷（TWP）可以降低糖尿病腎病24小時(shí)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(hù)作用，激活素A（Act-A）在糖尿病腎病中具有誘導(dǎo)腎小管間質(zhì)上皮細(xì)胞轉(zhuǎn)分化的作用，同時(shí)能促進(jìn)腎臟發(fā)生纖維化，通過(guò)下調(diào)激活素A（Act-A）的表達(dá)，從而阻礙腎小管間質(zhì)上皮細(xì)胞的轉(zhuǎn)分化，延緩腎臟發(fā)生纖維化的進(jìn)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(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R587.2;R692

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