本文選題：α-平滑肌肌動蛋白　切入點：輸尿管梗阻　出處：《武漢大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：腎間質(zhì)纖維化是多種進(jìn)展性腎臟疾病終末階段共同的病理表現(xiàn),其病理特征為：腎間質(zhì)炎性細(xì)胞浸潤、聚集,肌成纖維細(xì)胞(MyoF)增殖、活化和進(jìn)行性的細(xì)胞外基質(zhì)(ECM)積聚等。JNK相關(guān)的亮氨酸拉鏈蛋白JLP是一種介導(dǎo)生物信號轉(zhuǎn)導(dǎo)及細(xì)胞內(nèi)分子囊泡轉(zhuǎn)運的重要支架蛋白。JLP在小鼠的多種器官組織中被發(fā)現(xiàn),如腦、肺、脾臟、睪丸等。本研究首次探討了JLP基因缺失對小鼠單側(cè)輸尿管梗阻腎病模型腎間質(zhì)纖維化的影響及其可能機(jī)制。第一部分支架蛋白JLP基因缺失對單側(cè)輸尿管梗阻小鼠腎間質(zhì)纖維化的影響目的觀察支架蛋白JLP基因缺失對單側(cè)輸尿管梗阻(UUO)小鼠腎間質(zhì)纖維化的影響,探討JLP在梗阻性腎病腎纖維化形成中的作用及可能機(jī)制。方法將jlp野生型jlp+/+)小鼠和jlp敲除(jlp-/-)小鼠分為4組：jlp+/+小鼠假手術(shù)組(jlp+/+-Sham)、jlp-/-小鼠假手術(shù)組(jlp-/--Sham)、jlp-/-小鼠模型組(jlp-/--UUO)和jlp-/-小鼠模型組(jlp-/--UUO),分別于術(shù)后7d和14d處死動物。Masson染色觀察腎間質(zhì)纖維化；免疫熒光、免疫組化及Western印跡觀察JLP在正常jlp+/+小鼠腎組織表達(dá)與分布；免疫組化法檢測腎組織α-平滑肌肌動蛋白(α-SMA)、Ⅰ型膠原(COL-Ⅰ)、Ⅲ型膠原(COL-Ⅲ)表達(dá)；Western印跡法檢測腎組織α-SMA、COL-Ⅰ、COL-Ⅲ蛋白表達(dá)。結(jié)果jlp+1+小鼠腎組織表達(dá)JLP,主要分布于腎小管。Masson染色顯示,與jlp+/+-UUO組比較,jlp-/--UUO組腎間質(zhì)膠原纖維明顯增加,纖維化程度明顯加重(P0.05)；免疫組化顯示,與jlp-/--UUO組比較,jlp-/--UUO組腎皮質(zhì)α-SMA、 COL-Ⅰ和COL-Ⅲ表達(dá)明顯升高(均P0.05);Western印跡顯示,jlp-/--UUO組α-SMA、COL-Ⅰ和COL-Ⅲ蛋白表達(dá)明顯升高(均P0.05)。結(jié)論支架蛋白JLP可抑制肌成纖維細(xì)胞的生成,在拮抗腎纖維化的形成中起到重要作用。第二部分支架蛋白JLP基因缺失對單側(cè)輸尿管梗阻小鼠腎間質(zhì)纖維化炎癥因子表達(dá)的影響目的觀察支架蛋白JLP基因缺失對UUO小鼠腎間質(zhì)巨噬細(xì)胞聚集、炎癥因子釋放的影響,探討JLP在梗阻性腎病腎纖維化形成中的作用。方法將ilp+/+小鼠和jlp-/-小鼠分為4組：jlp+/+-Sham組、jlp-/--Sham組、jlp+/+-UUO組和jlp-/--UUO組,于術(shù)后7d處死動物。實時熒光定量PCR檢測腎組織腫瘤壞死因子-α(TNF-α)、白細(xì)胞介素-1p(IL-1p)和單核細(xì)胞趨化蛋白-1(MCP-1)mRNA的表達(dá)；免疫組化法檢測腎組織巨噬細(xì)胞標(biāo)志物CD68、炎癥因子TNF-α、IL-1β和MCP-1的表達(dá)；Western印跡檢測腎組織TNF-α、IL-1β和MCP-1蛋白的表達(dá)。結(jié)果實時熒光定量PCR顯示,jlp/-/-UUO組TNF-α、IL-1β和MCP-1mRNA表達(dá)水平明顯升高,與jlp+/+-UUO組比較,差異有統(tǒng)計學(xué)意義(均P0.05)；免疫組化顯示,與jlp+/+-UUO組比較,jlp-/--UUO組腎皮質(zhì)CD68、TNF-α、IL-1β和MCP-1表達(dá)明顯升高(均P0.05)；同時,Western印跡顯示,jlp-/--UUO組TNF-α、IL-1β和MCP-1蛋白表達(dá)明顯升高(均P0.05)結(jié)論支架蛋白JLP在抑制梗阻性腎病腎間質(zhì)巨噬細(xì)胞聚集和炎癥因子的釋放中起到重要作用。第三部分支架蛋白,ILP基因缺失對單側(cè)輸尿管梗阻小鼠腎臟TGF-β1/Smad信號通路及細(xì)胞凋亡的影響目的觀察支架蛋白JLP基因缺失對UUO小鼠腎臟TGF-β1/Smad及腎小管間質(zhì)細(xì)胞凋亡的影響,探討JLP在梗阻性腎病腎纖維化形成中可能機(jī)制。方法將jlp+/+小鼠和jlp-/-小鼠分為4組：jlp+/+-Sham組、jlp-/--Sham組、jlp+/+-UUO組和jlp-/--UUO組,分別于術(shù)后7d和14d處死動物。實時熒光定量PCR檢測腎組織轉(zhuǎn)化生長因子β1(TGF-β1)mRNA的表達(dá)；免疫組化法檢測腎組織TGF-β1的表達(dá)；Western印跡檢測腎組織TGF-β1、p-Smad2、p-Smad3及p-P65蛋白的表達(dá)；TUNEL法檢測腎臟間質(zhì)細(xì)胞凋亡。結(jié)果實時熒光定量PCR顯示,jlp-/--UUO組TGF-β1mRNA表達(dá)水平明顯升高,與jlp+/+-UUO組比較,差異有統(tǒng)計學(xué)意義(P0.05)；免疫組化顯示,與jlp+/+-UUO組比較,jlp-/--UUO組腎皮質(zhì)TGF-β1表達(dá)明顯升高(P0.05)；同時,Western印跡顯示,jlp-/--UUO組TGF-β1、p-Smad2、p-Smad3及p-P65蛋白表達(dá)明顯升高(均P0.05)；TUNEL顯示,與jlp+/+-UUO組比較,jlp-/--UUO組腎臟間質(zhì)細(xì)胞凋亡率明顯升高(P0.05)。結(jié)論支架蛋白JLP可抑制TGF-β1/Smad信號通路及NF-κB信號通路的激活、并減少細(xì)胞凋亡,在拮抗腎纖維化的形成中起重要作用。
[Abstract]:Renal interstitial fibrosis is a pathologic variety of progressive renal disease, end stage common manifestations, the pathological features of renal interstitial infiltration of inflammatory cells, aggregation, fibroblast (MyoF) proliferation, activation and extracellular matrix (ECM) accumulation of.JNK related leucine zipper protein JLP is a kind of important biological scaffold protein.JLP mediated signal transduction and cellular vesicular transport has been found in a variety of organs and tissues in mice, such as brain, lung, spleen, testis and so on. This study is the first to investigate the effects of renal JLP gene deletion on model mice with unilateral ureteral obstruction renal interstitial fibrosis and its possible mechanism the first part. The scaffold protein JLP gene deletion in mice with unilateral ureteral obstruction on renal interstitial fibrosis Objective To observe the effects of scaffold protein JLP gene deletion in unilateral ureteral obstruction (UUO) mice renal interstitial fibrosis, explore JLP In renal fibrosis in obstructive nephropathy in the formation of the effects and the possible mechanism. Methods: JLP wild type jlp+/+) mice and JLP knockout (jlp-/-) mice were divided into 4 groups: sham operation group jlp+/+ mice (jlp+/+-Sham mice), jlp-/- sham operation group (jlp-/--Sham), jlp-/- model group (jlp-/--UUO) and jlp-/- mice model group (jlp-/--UUO), respectively after 7d and 14d were sacrificed animal.Masson staining to observe the renal interstitial fibrosis; immunofluorescence, immunohistochemistry and Western blotting to observe the expression and distribution of JLP in renal tissue of normal jlp+/+ mice; immunohistochemistry in renal tissue of alpha smooth muscle actin (alpha -SMA), collagen (COL- I), collagen type III (COL- III) expression in renal tissue was detected by Western blotting; alpha -SMA, COL- 1, COL- expression protein. Results the expression of JLP in renal tissue of jlp+1+ mice, mainly in renal tubular.Masson staining showed that compared with jlp+/+-UUO group, jlp-/--U UO group of renal interstitial collagen fibers increased significantly, the degree of fibrosis was significantly increased (P0.05); immunohistochemistry showed that, compared with jlp-/--UUO group, jlp-/--UUO group of renal cortical alpha -SMA, expression of COL- I and COL- III were significantly increased (P0.05); Western blot showed that jlp-/--UUO group, alpha -SMA, COL- I and COL- III protein expression increased significantly (P0.05). Conclusion the scaffold protein JLP can inhibit the generation of muscle fiber cells, play an important role in the formation of anti renal fibrosis. Some of the scaffold protein JLP gene deletion second on expression of inflammatory factor of fiber in mice with unilateral ureteral obstruction kidney to observe the scaffold protein JLP gene deletion in UUO mice renal interstitial macrophages, inflammatory factors release, formation of JLP in renal fibrosis in obstructive nephropathy in rats. Methods ilp+/+ mice and jlp-/- mice were divided into 4 groups: jlp+/+ -Sham group, jlp-/--Sha M group, jlp+/+-UUO group and jlp-/--UUO group, sacrificed on 7d animal kidney tissue. Real time fluorescence quantitative PCR detection of tumor necrosis factor alpha (TNF- alpha), interleukin -1p (IL-1p) and monocyte chemoattractant protein -1 (MCP-1) mRNA expression; immunohistochemistry in renal tissue of giant macrophage marker CD68, inflammatory factor TNF- alpha, IL-1 beta and MCP-1 expression; Western blot detection of TNF- expression in renal tissue of alpha, IL-1 beta and MCP-1 protein. Results of real-time PCR showed that jlp/-/-UUO group TNF- alpha, IL-1 beta and MCP-1mRNA expression levels were significantly increased, compared with jlp+/+-UUO group, the difference was statistically significant (P0.05); immunohistochemistry showed that, compared with jlp+/+-UUO group, jlp-/--UUO group, renal cortex CD68, TNF- alpha, IL-1 beta and MCP-1 expression increased significantly (P0.05); at the same time, Western blotting showed that the jlp-/--UUO group of TNF- alpha, IL-1 beta and MCP-1 protein expression increased significantly (P0.05) conclusion Play an important role in the inhibition of the scaffold protein JLP in obstructive nephropathy renal interstitial macrophage accumulation and release of inflammatory factors. The third part scaffold protein, the effect of ILP gene deletion on mouse kidney with unilateral ureteral obstruction TGF- beta 1/Smad signaling pathway and apoptosis to observe support protein JLP gene deletion effect on UUO mouse kidney TGF- beta 1/Smad and tubulointerstitial cell apoptosis, explore the formation of JLP in renal fibrosis in obstructive nephropathy in the possible mechanism. Methods: jlp+/+ mice and jlp-/- mice were divided into 4 groups: jlp+/+-Sham group, jlp-/--Sham group, jlp+/+-UUO group and jlp-/--UUO group, respectively after 7d and 14d were animal. Real time fluorescence quantitative PCR detection of renal tissue transformation growth factor beta 1 (TGF- beta 1) mRNA expression; immunohistochemistry was used to detect the expression of TGF- in renal tissue beta 1; renal tissue Western blot detection of TGF- beta 1, p-Smad2, p-Smad3 and p-P65 protein The expression of TUNEL was detected; renal interstitial cell apoptosis. Results of real-time PCR showed that the expression of jlp-/--UUO in group TGF- beta 1mRNA levels were significantly increased, compared with jlp+/+-UUO group, the difference was statistically significant (P0.05); immunohistochemistry showed that, compared with jlp+/+-UUO group, jlp-/ --UUO group of renal cortical TGF- expression was significantly increased (p 1 P0.05); at the same time, Western blotting showed that jlp-/--UUO group TGF- beta 1, p-Smad2, p-Smad3 and the expression of p-P65 protein increased significantly (P0.05); TUNEL showed that, compared with jlp+/+-UUO group, jlp-/--UUO group of renal interstitial cell apoptosis rate increased significantly (P0.05). Conclusion: the activation of the scaffold protein JLP can inhibit the 1/Smad signaling pathway and TGF- NF- B signaling pathway, and reduce apoptosis, play an important role in the formation of anti renal fibrosis.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R692

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