本文關(guān)鍵詞： Necrostatin-1受體相互作用蛋白1 受體相互作用蛋白3 缺血再灌注損傷 程序性壞死　出處：《天津醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的：通過建立大鼠腎缺血再灌注損傷(Ischemia-Reperfusion Injury, IRI)模型,觀察其對大鼠腎病理、生化指標(biāo)、炎癥因子及細(xì)胞程序性壞死的影響,并觀察Necrostatin-1(Nec-1)對腎的保護(hù)作用,探討其可能的機(jī)制,為臨床治療腎缺血再灌注損傷提供理論依據(jù)。 方法：將90只雄性SD大鼠按隨機(jī)數(shù)字表法分為3組：假手術(shù)組、DMSO對照組、Nec-1組,每組30只；每組按2h、12h、24h再分為3組,每組10只。DMSO對照組和Nec-1組采用夾閉雙側(cè)大鼠腎動脈45min再恢復(fù)血流的方法制成腎缺血再灌注模型,于模型完成前15min尾靜脈給藥。假手術(shù)組不夾閉雙側(cè)腎動脈。Nec-1組于再灌15min前尾靜脈注射Nec-11mg/kg(由DMSO容解),DMSO對照組注射等體積DMSO,假手術(shù)組只游離雙腎,不作任何處理。模型成功后收集血清及腎臟組織；檢測血清肌酐(serum creatinine, Scr).血尿素氮(blood urea nitrogen, BUN);HE染色觀察腎組織病理改變并計算腎小管壞死評分；酶聯(lián)免疫吸附試驗(enzyme linked immunosorbent assay, ELISA)方法檢測腫瘤壞死因子-α (TNF-α)白細(xì)胞介素-1p(IL-1p)濃度；Western blot方法檢測受體相互作用蛋白1和3(RIP1,RIP3)的表達(dá)。 結(jié)果：1.與假手術(shù)組相比,DMSO對照組及Nec-1組大鼠各時間點Scr、BUN水平均升高(P0.01),以DMSO組升高更為明顯。隨著時間推移,兩組Scr、BUN水平均升高,Nec-1組大鼠各時間點Scr、BUN水平比DMSO對照組均降低(P0.05)。 2.假手術(shù)組大鼠腎組織結(jié)構(gòu)清晰,腎小管上皮細(xì)胞完整,排列整齊。DMSO對照組腎小管結(jié)構(gòu)異常,細(xì)胞脫落壞死、腫脹、管型形成、炎細(xì)胞浸潤明顯。與DMSO對照組相比,Nec-1組細(xì)胞脫落壞死、腫脹、管型形成均減輕,炎細(xì)胞相對減少。與假手術(shù)組相比,DMSO組與Nec-1組大鼠各時間點腎小管壞死死評分均明顯升高(P0.01)以DMSO組升高更為顯著。隨著時間推移,兩組Scr. BUN水平均升高,Nec-1組大鼠各時間點腎小管壞死評分水平比DMSO對照組均降低(P0.05)。 3.DMSO對照組及Nec-1組大鼠各時間點TNF-α、IL-1β表達(dá)比假手術(shù)組明顯升高(P0.01),以DMSO組升高更為明顯。隨著時間推移,兩組TNF-α、IL-1β水平均升高,Nec-1組大鼠各時間點TNF-α、IL-1β比DMSO對照組均降低(P0.01)。 4.假手術(shù)組RIP1和RIP3蛋白的表達(dá)量很低。與假手術(shù)組相比,DMSO對照組及Nec-1組RIP1和RIP3蛋白從2h開始就有較明顯表且的表達(dá),且隨時間升高(P0.01)。隨著時間推移,兩組RIP1和RIP3蛋白水平均升高,與DMSO對照組相比,Nec-1組RIP1和RIP3蛋白的表達(dá)差異無統(tǒng)計學(xué)意義(P0.05)。 結(jié)論：1.腎缺血再灌注損傷可導(dǎo)致大鼠腎組織結(jié)構(gòu)破壞,細(xì)胞脫落壞死,腎小管壞死評分明顯升高,反應(yīng)腎功能水平的Scr、BUN水平均升高。Nec-1能有效減輕腎缺血再灌注損傷,改善腎功能。 2.腎缺血再灌注損傷可激活大鼠免疫反應(yīng),引起TNF-α、IL-1β等炎癥因子明顯升高。Nec-1能有效抑制免疫炎癥反應(yīng),減輕損傷。 3.腎缺血再灌注損傷可引起嚴(yán)重的程序性壞死,RIP1和RIP3蛋白升高明顯。Nec-1能抑制RIP1和RIP3蛋白活性,減輕程序性壞死對腎組織損傷程度。 4.Nec-1能明顯改善大鼠腎缺血再灌注損傷組織破壞、炎癥反應(yīng),減少程序性壞死的發(fā)生,其機(jī)制可能與抑制細(xì)胞程序性壞死有關(guān)。
[Abstract]:Objective: to establish the rat renal ischemia reperfusion injury (Ischemia-Reperfusion, Injury, IRI) model, observe the biochemical indicators of renal pathology, rats, inflammatory factors and programmed cell necrosis Necrostatin-1 (Nec-1), and to observe the protective effect on the kidney, to explore the possible mechanism for the clinical treatment of renal ischemia reperfusion injury and provide a theoretical basis.
Methods: 90 male SD rats were randomly divided into 3 groups: sham operation group, DMSO control group, Nec-1 group, 30 rats in each group; each group according to the 2H, 12h, 24h was subdivided into 3 groups, 10 rats in each group.DMSO control group and Nec-1 group by clamping bilateral renal artery of 45min rats to restore blood flow by renal ischemia reperfusion model, before the completion of 15min intravenous injection in the model. The sham operation group without occlusion of bilateral renal artery in.Nec-1 group after 15min reperfusion before intravenous injection of Nec-11mg/kg (DMSO, DMSO solution containing) control group was injected with equal volume of DMSO, the sham operation group only free double kidney, without any treatment. Serum and kidney tissue after successful model; detection of serum creatinine (serum creatinine, Scr). Blood urea nitrogen (blood urea, nitrogen, BUN); HE staining was used to observe the pathological change of kidney tissue and renal tubular necrosis score; enzyme linked immunosorbent assay (enzyme linked immunosorbent as Say (ELISA) method was used to detect the concentration of tumor necrosis factor alpha (TNF- alpha) interleukin -1p (IL-1p). Western blot method was used to detect the expression of receptor interacting protein 1 and 3 (RIP1, RIP3).
Results: 1. compared with sham operation group, DMSO control group and Nec-1 group at different time points in rats Scr, BUN levels were significantly increased (P0.01), in DMSO group increased more significantly. With the passage of time, two groups of Scr, BUN levels were elevated, Nec-1 rats at each time point Scr, BUN level than DMSO the control group were lower (P0.05).
2. sham operation group rats renal tissue structure is clear, renal tubular epithelial cells, arranged in.DMSO group of renal tubular abnormalities, exfoliative cell necrosis, swelling, tube formation, inflammatory cell infiltration. Compared with DMSO control group, Nec-1 group of exfoliated cells necrosis, swelling and tube formation were reduced, inflammatory cells relatively reduced. Compared with sham operation group, DMSO group and Nec-1 group at different time points in rat renal tubular and bad scores were significantly increased (P0.01) in DMSO group increased more significantly. With the passage of time, Scr. BUN levels were increased in all two groups, each time point of renal tubular necrosis in rats in group Nec-1 were better than DMSO the control group were lower (P0.05).
Each time point 3.DMSO control group and Nec-1 group rats TNF- alpha, IL-1 beta expression was significantly higher than that of sham operation group (P0.01), in DMSO group increased more significantly. With the passage of time, two groups of TNF- alpha, IL-1 beta levels were elevated, each time point TNF- alpha Nec-1 rats, IL-1 beta DMSO the control group were lower (P0.01).
The expression of RIP1 4. in sham operation group and RIP3 protein is very low. Compared with sham operation group, DMSO group and Nec-1 group of RIP1 and RIP3 protein 2H from the start and has obvious expression, and increased with time (P0.01). With the passage of time, two groups of RIP1 and RIP3 protein levels were elevated. Compared with DMSO control group, no statistically significant difference between group RIP1 and the expression of Nec-1 RIP3 protein (P0.05).
Conclusion: 1., renal ischemia-reperfusion injury can lead to the destruction of renal structure, cell loss and necrosis, and the increase of renal tubular necrosis. The level of Scr and BUN in response to renal function is increased..Nec-1 can effectively alleviate renal ischemia-reperfusion injury and improve renal function.
2., renal ischemia-reperfusion injury can activate the immune response in rats, induce TNF-, IL-1 and other inflammatory factors to increase significantly,.Nec-1 can effectively inhibit immune inflammatory response and reduce injury.
3., renal ischemia-reperfusion injury can cause severe procedural necrosis. RIP1 and RIP3 protein increase..Nec-1 can inhibit the activity of RIP1 and RIP3 protein, and reduce the degree of renal damage in programmed necrosis.
4.Nec-1 can obviously improve the tissue destruction, inflammatory reaction and reduce the occurrence of programmed necrosis in rats with renal ischemia-reperfusion injury. The mechanism may be related to the inhibition of programmed cell death.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R699.2

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