本文選題：腎缺血再灌注損傷　切入點：萊菔硫烷　出處：《河北醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：腎缺血再灌注損傷(Renal ischemia reperfusion injury,RIRI)是臨床上一種常見的病理生理現(xiàn)象,多見于腎單位保留術(shù)、腎擠壓傷、腎動脈阻斷術(shù)以及腎臟切除或移植等臨床治療過程中。大量動物實驗和臨床研究均發(fā)現(xiàn):在暫時性阻斷腎臟的血液灌注,隨后又重新恢復(fù)其血液供應(yīng)的過程中,會有大量活性氧族(reac-tive oxygen species,ROS)產(chǎn)生,導(dǎo)致腎組織細(xì)胞的嚴(yán)重?fù)p傷。因此,氧化應(yīng)激被認(rèn)為是引發(fā)缺血再灌注損傷的主要機(jī)制之一。核因子E2相關(guān)因子2(nuclear factor erythroid 2-related factor 2,Nrf2)是目前發(fā)現(xiàn)的最強(qiáng)抗氧化應(yīng)激轉(zhuǎn)錄調(diào)控因子。Nrf2通過與抗氧化反應(yīng)元件(antioxidant responsive element,ARE)相互作用調(diào)節(jié)抗氧化蛋白的表達(dá),是機(jī)體內(nèi)重要的內(nèi)源性抗氧化應(yīng)激通路。超氧化物歧化酶(Superoxide Dismutases,SOD)是機(jī)體內(nèi)主要的ROS清除酶系,能通過歧化反應(yīng)將羥自由基(OH-)轉(zhuǎn)化成氧。同時,SOD也是Nrf2轉(zhuǎn)錄調(diào)控的下游靶基因之一。萊菔硫烷(sulforaphane,SFN)在西蘭花等十字花科蔬菜中大量存在,屬于異硫氰酸鹽。大量研究表明:SFN是Nrf2通路的誘導(dǎo)劑,可通過上調(diào)Nrf2及其下游基因的表達(dá)發(fā)揮抗氧化、抗腫瘤等生物學(xué)作用。本研究采用無損傷動脈夾鉗夾腎動脈法建立大鼠RIRI模型,同時給予RIRI模型組大鼠Nrf2誘導(dǎo)劑萊菔硫烷,觀察萊菔硫烷處理的大鼠RIRI模型腎組織形態(tài)學(xué)及功能變化、H_2O_2含量、MDA含量及抗氧化蛋白SOD活性及表達(dá)變化,探討萊菔硫烷在RIRI中發(fā)揮的抗氧化作用。目的:通過觀察萊菔硫烷處理的大鼠RIRI模型腎組織形態(tài)學(xué)和功能變化、H_2O_2含量、MDA含量及抗氧化蛋白SOD活性及表達(dá)變化,探討萊菔硫烷是否可通過上調(diào)Nrf2下游基因SOD的表達(dá),增強(qiáng)機(jī)體對ROS的清除作用,降低腎組織過氧化損傷程度,為萊菔硫烷對RIRI的防治研究提供數(shù)據(jù)依據(jù)。方法:1動物模型建立及檢測標(biāo)本的處理Wistar大鼠來源于河北醫(yī)科大學(xué)實驗動物中心,雄性(體重200±10g)24只,隨機(jī)分為對照組(Control組)、腎臟缺血再灌注損傷模型組(RIRI組)、缺血+萊菔硫烷組(SFN1組),灌注+萊菔硫烷組(SFN2組)。按照余曉東等人的模型制備方法建立RIRI實驗動物模型:腹腔注射6%水合氯醛(5ml/kg)麻醉大鼠。將RIRI組大鼠固定于手術(shù)臺上,酒精消毒后開腹充分暴露雙側(cè)腎臟,先將大鼠的右側(cè)腎臟切除后,再鈍性地分離其左側(cè)腎動脈,并采用無創(chuàng)傷性動脈夾在接近腎門部位夾閉左側(cè)腎動脈,阻斷腎臟血液供應(yīng),此時可以觀察到腎臟顏色由鮮紅色迅速轉(zhuǎn)變?yōu)榘导t色。血液灌注阻斷45分鐘后棄去動脈夾,重新恢復(fù)左側(cè)腎臟的血液灌注,此時可見左側(cè)腎動脈迅速充盈,腎臟顏色也隨即由暗紅色轉(zhuǎn)變?yōu)轷r紅色,提示左側(cè)腎臟的血液重新灌注成功。Control組大鼠的消毒開腹過程與RIRI模型組一致,但該組大鼠雙側(cè)腎臟暴露后只切除其右側(cè)腎臟,鈍性地分離左側(cè)腎動脈,但并不夾閉阻斷血液灌注。SFN1組大鼠在夾閉左側(cè)腎動脈后立即將二甲基亞砜稀釋的萊菔硫烷均勻涂抹于小腸表面,其余操作步驟同RIRI組。SFN2組大鼠在棄去血管夾恢復(fù)腎臟血液灌注后立即將二甲基亞砜稀釋的萊菔硫烷均勻涂抹于小腸表面,其余操作步驟同RIRI組。Control組和RIRI組只在小腸表面涂抹等量的二甲基亞砜�；謴�(fù)大鼠腎臟血流灌注24小時后重新麻醉大鼠,收集右頸總動脈血液,3000轉(zhuǎn)/分,離心10分鐘,分離并收集血清,用于血清肌酐(Serum Creatinine,SCr)、血清尿素氮(Blood Urea Nitrogen,BUN)含量檢測;大鼠處死后切取其腎臟,取適宜大小的腎臟標(biāo)本置于4%多聚甲醛固定液中固定,用于HE染色法觀察大鼠腎臟組織的形態(tài)學(xué)改變。將其余腎臟組織迅速置于液氮中,待冷凍后轉(zhuǎn)移至-80℃冰箱保存?zhèn)溆?用于大鼠腎組織SOD活性、基因表達(dá)水平測定,丙二醛(malondialdehyde,MDA)含量和過氧化氫(hydrogen peroxide,H_2O_2)含量測定。2檢測指標(biāo)及測定方法2.1大鼠血清內(nèi)SCr和BUN含量檢測采用苦味酸法檢測大鼠血清SCr含量;采用酶偶聯(lián)速率法測定大鼠血清BUN含量。SCr和BUN含量的測定過程嚴(yán)格按照試劑盒操作說明進(jìn)行。2.2大鼠腎臟組織形態(tài)結(jié)構(gòu)改變的觀察采用HE染色法觀察腎臟組織形態(tài)結(jié)構(gòu)的改變。將4%多聚甲醛固定的腎臟標(biāo)本按梯度酒精脫水、二甲苯透明,石蠟包埋處理后,將腎臟標(biāo)本切片厚度約5μm,然后進(jìn)行蘇木精伊紅染色處理,采用奧林帕斯光學(xué)顯微鏡觀察腎組織形態(tài)學(xué)改變并拍照。2.3大鼠腎組織MDA含量檢測腎組織MDA含量采用硫代巴比妥酸比色法測定,檢測過程按試劑盒操作說明進(jìn)行。2.4大鼠腎組織H_2O_2含量檢測腎組織H_2O_2含量采用鉬酸比色法測定,檢測過程按試劑盒操作說明進(jìn)行。2.5大鼠腎組織SOD活性檢測腎組織SOD活性采用黃嘌呤氧化酶法測定,檢測過程按試劑盒操作說明進(jìn)行2.6大鼠腎組織SOD基因表達(dá)水平檢測采用RNA提取試劑盒裂解提取大鼠腎組織內(nèi)的總RNA。RNA定量后將2μg RNA反轉(zhuǎn)錄成模板c DNA。以甘油醛-3-磷酸脫氫酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)作為內(nèi)參照,進(jìn)行Real-Time PCR相對定量分析。結(jié)果:1大鼠腎組織的HE觀察結(jié)果HE法觀察大鼠腎組織形態(tài)結(jié)構(gòu)改變。光鏡下可見:Control組大鼠腎近曲小管、遠(yuǎn)曲小管形態(tài)結(jié)構(gòu)清晰完整。腎小球形態(tài)規(guī)則、腎小囊大小正常。RIRI組大鼠:腎小球明顯萎縮體積變小,腎小囊間隙顯著增寬,近曲小管、遠(yuǎn)曲小管細(xì)胞萎縮,胞質(zhì)內(nèi)可見空泡,管腔擴(kuò)張明顯。SFN2組大鼠:腎小球略有萎縮體積變小嗜伊紅染色增強(qiáng),腎小囊有所顯擴(kuò)張,近曲小管細(xì)胞略顯腫脹,小管之間間隙變窄,遠(yuǎn)曲小管管腔增寬。SFN1組大鼠:腎小球嗜伊紅染色有所增強(qiáng),腎小囊間隙略顯增寬,近曲小管略有腫脹,小管之間間隙變窄,遠(yuǎn)曲小管管腔略增寬程度輕于SFN2組。2血清內(nèi)SCr含量的改變與Control組大鼠血清內(nèi)SCr的含量(59.87±6.76μmol/L)相比,RIRI組(358.08±22.20μmol/L)、SFN2組(294.14±18.76μmol/L)、SFN1組(93.60±13.16μmol/L)大鼠血清SCr含量均明顯升高(P0.05),但SFN2組和SFN1組大鼠血清SCr含量低于RIRI組(P0.05),SFN1組大鼠血清SCr含量又低于SFN2組(P0.05)。3血清內(nèi)BUN含量的改變與Control組大鼠血清內(nèi)BUN的含量(6.35±1.15 mmol/L)相比,RIRI組(24.80±2.53 mmol/L)、SFN2組(20.01±1.78 mmol/L)、SFN1組(12.32±1.69mmol/L)大鼠血清BUN含量均明顯升高(P0.05),但SFN2組和SFN1組大鼠血清BUN含量低于RIRI組(P0.05),SFN1組大鼠血清BUN含量又低于SFN2組(P0.05)。4腎組織MDA含量的改變與Control組大鼠腎組織MDA含量(72.29±14.75 mmol/g)相比,RIRI組(284.32±17.48 mmol/g)、SFN2組(186.52±17.50 mmol/g)、SFN1組(161.31±14.03 mmol/g)大鼠腎組織MDA含量均明顯升高(P0.05),但SFN2組和SFN1組大鼠腎組織MDA含量明顯低于RIRI組(P0.05),SFN1組大鼠腎組織MDA含量又低于SFN2組(P0.05)。5腎組織H_2O_2含量的改變與Control組大鼠腎組織H_2O_2含量(85.70±10.66 mmol/g)相比,RIRI組(260.62±48.50 mmol/g)、SFN2組(160.84±23.34 mmol/g)、SFN1組(121.22±18.49 mmol/g)大鼠腎組織H_2O_2含量均明顯升高(P0.05),但SFN2組和SFN1組大鼠腎組織H_2O_2含量明顯低于RIRI組(P0.05),SFN1組大鼠腎組織H_2O_2含量又低于SFN2組(P0.05)。6大鼠腎組織SOD活性改變與Control組大鼠腎組織SOD活性(112.47±9.09 U/mg pro)相比,RIRI組(28.56±6.11 U/mg pro)、SFN2組(43.85±5.29 U/mg pro)、SFN1組(65.44±8.91 U/mg pro)大鼠腎組織SOD活性均明顯降低(P0.05),但SFN2組和SFN1組大鼠腎組織SOD活性高于RIRI組(P0.05),SFN1組大鼠腎組織SOD活性又高于SFN2組(P0.05)。7腎組織SOD基因的表達(dá)改變Real-Time PCR法測定大鼠腎組織SOD的m RNA相對表達(dá)水平,GAPDH作為擴(kuò)增內(nèi)對照。與Control組大鼠腎組織SOD m RNA表達(dá)量(0.93±0.13)相比,RIRI組(1.15±0.15)、SFN2組(1.36±0.18)、SFN1組(1.39±0.18)大鼠腎組織SOD m RNA表達(dá)水平均升高(P0.05)。SFN2組和SFN1組大鼠腎組織SOD的m RNA相對表達(dá)水平又高于RIRI組(P0.05),但SFN2組和SFN1組SOD的m RNA相對表達(dá)水平無明顯差異(P㧐0.05)。此結(jié)果表明:在RIRI發(fā)生過程中,SFN上調(diào)了SOD基因表達(dá)水平。結(jié)論:1腎臟缺血再灌注損傷發(fā)生后,腎組織處于氧化應(yīng)激狀態(tài)并遭受過氧化損傷。2腎臟缺血再灌注損傷發(fā)生后,萊菔硫烷通過上調(diào)Nrf2下游基因SOD的表達(dá),增強(qiáng)機(jī)體對ROS的清除作用,降低了腎組織的過氧化損傷程度。3與再灌注后給藥相比,缺血后即刻給予萊菔硫烷更能有效降低腎組織氧化應(yīng)激水平和過氧化損傷程度,改善腎臟的形態(tài)結(jié)構(gòu)和功能改變。
[Abstract]:Renal ischemia reperfusion injury (Renal ischemia reperfusion injury, RIRI) is a common clinical pathological and physiological phenomenon in nephron sparing surgery, renal crush injury, renal artery occlusion and renal resection or transplantation in the clinical treatment process. A large number of animal experiments and clinical studies have found that in the temporary occlusion of blood renal perfusion, then returned to the blood supply, there will be a large number of reactive oxygen species (reac-tive oxygen, species, ROS), causing serious damage of renal tissue cells. Therefore, oxidative stress is considered to be one of the main causes of ischemia-reperfusion injury. Nuclear factor E2 related factor 2 (nuclear factor erythroid 2-related factor 2, Nrf2.Nrf2) is currently the strongest antioxidant stress transcription factor found with antioxidant response element (antioxidant, responsive element, ARE) interaction adjustment Section expression of antioxidant proteins is an important endogenous antioxidant stress pathway. Superoxide dismutase (Superoxide Dismutases SOD) is the main body of the ROS scavenging enzyme system, the hydroxyl radical by disproportionation (OH-) into oxygen. At the same time, one of the downstream target gene SOD and Nrf2 transcription. Sulforaphane (sulforaphane, SFN) in the presence of a large number of broccoli and other cruciferous vegetables, which belongs to the isothiocyanates. Many studies show that SFN is the inducer of the Nrf2 pathway, through the upregulation of the expression of Nrf2 and its downstream genes play anti oxidation, anti tumor biological effects. This study uses non-invasive artery clamp renal artery clamping method to establish a rat model of RIRI, given the RIRI model rats induced by Nrf2 agent sulforaphane, observe the changes of morphology and function in renal tissue of RIRI rat model of sulforaphane treated H_2O_2 content, MDA content and The changes and expression of antioxidant protein SOD activity, to explore the antioxidant effects of sulforaphane play in RIRI. Objective: To observe the changes of morphology and function in renal tissue of RIRI rat model of sulforaphane treatment H_2O_2 content and expression of MDA SOD protein content and antioxidant activity of sulforaphane, whether through upregulation of the expression of Nrf2 the downstream gene of SOD, enhanced the scavenging effect of ROS, reduce the renal oxidative damage degree, provide data basis for the study of sulforaphane on prevention and treatment of RIRI. Methods: 1 animal models and specimens and Wistar rats from the experimental animal center of Hebei Medical University, male (weight 200 + 10g) 24 were randomly divided into control group (Control group), model group renal ischemia reperfusion injury (RIRI group), ischemia + sulforaphane group (SFN1 group), reperfusion + sulforaphane group (SFN2 group) according to Yu Xiaodong et al. The preparation method of the RIRI model to establish an experimental animal model: intraperitoneal injection of 6% chloral hydrate (5ml/kg) in anesthetized rats. The rats in RIRI group were fixed on the operating table, alcohol disinfection after laparotomy fully exposed bilateral kidneys, the right kidney of rats after resection, and blunt separation of the left renal artery, and the clip near the hilum part clamping left renal artery without traumatic occlusion of renal artery, blood supply, this can be observed in the kidney color quickly changed from bright red to dark red. The blood perfusion after occlusion for 45 minutes and discard the artery clamp, restoring blood perfusion of the left kidney, this is visible on the left renal artery rapidly filling kidney the color was dark red shift by bright red, suggesting that the left kidney blood reperfusion success in rats of.Control group and RIRI model group disinfectionand operation process, but the rats exposed only after resection of bilateral kidneys The right kidney, blunt separation of left renal artery occlusion, but not blocking the blood perfusion of rats in the.SFN1 group in the clamping of the left renal artery immediately after sulforaphane two DMSO diluted evenly on the surface of the small intestine, the remaining steps with the RIRI group.SFN2 group of rats in the blood discarding recovery pipe clamp renal blood perfusion immediately after sulforaphane two DMSO diluted evenly on the surface of the small intestine, two DMSO remaining steps with the RIRI group.Control group and RIRI group only apply in the small intestine. The equivalent surface recovery of rat kidney perfusion again after 24 hours of anesthesia in rats, the right carotid artery blood collection 3000, RPM, centrifugal separation for 10 minutes, and serum was collected for serum creatinine (Serum Creatinine, SCr), blood urea nitrogen (Blood Urea, Nitrogen, BUN) were detected; the rats were killed after excision of the kidney, and suitable for small samples of kidney structure In 4% paraformaldehyde fixative, used to observe the morphological changes of renal tissue in rats by HE staining. The kidney tissue rapidly in liquid nitrogen, to be frozen after transfer to -80 deg.c refrigerator spare, for the activity of SOD in rat renal tissue, to determine the level of gene expression, C two aldehydes (malondialdehyde, MDA) and the content of hydrogen peroxide (hydrogen peroxide, H_2O_2) determination of.2 detection index and the method for the determination of 2.1 rat serum SCr and BUN were detected by picric acid method to detect serum SCr content in rats; process of determination of rat serum BUN content of.SCr and BUN was determined by enzyme coupling rate method in strict accordance with the kit instructions were observed. The change of.2.2 in rat kidney tissue morphology observed by HE morphology of kidney tissue staining. The change of poly 4% kidneys were fixed in formaldehyde dehydrated by gradient alcohol, toluene two transparent, Paraffin embedded renal tissue sections after the treatment, the thickness of about 5 m, and then by hematoxylin eosin staining, using Olympus optical microscope to observe the morphological changes of the kidney tissue was determined by colorimetric method of thiobarbituric acid photo MDA content of renal tissue to detect the content of MDA in renal tissue of.2.3 rats, the detection process according to the kit instructions than the molybdate colorimetric method for determination of H_2O_2 content of renal tissue to detect the content of H_2O_2 in renal tissue of.2.4 rats, the detection process according to the kit instructions for the SOD activity of renal tissue to detect the activity of SOD in renal tissue of.2.5 rats was determined by xanthine oxidase method, the detection process according to the kit instructions for renal SOD gene expression of rats was detected by 2.6 RNA extraction kit to extract total RNA.RNA cracking quantitative renal tissue in rats after 2 g RNA reverse transcription template C DNA. with glyceraldehyde dehydrogenase (Gly -3- Ceraldehyde-3-phosphate dehydrogenase, GAPDH) as internal reference, Real-Time PCR relative quantitative analysis. Results: 1 rat renal tissue HE results HE method was used to observe the morphology of rat kidney. Under light microscope: in Control group of renal proximal tubules, distal convoluted tubule structure is clear and complete. Glomerular morphology rules. The renal capsule size of normal rats in the.RIRI group: glomerular atrophy volume, renal capsule gap widened significantly, proximal tubules, distal convoluted tubule atrophy, intracytoplasmic vacuoles, dilated obviously in.SFN2 rats: glomerular shrink slightly smaller eosinophilic staining enhancement, renal capsule has significant expansion the proximal tubule cells, slightly swollen, narrow tubules between the distal tubule lumen widened.SFN1 rats glomerular eosinophilic staining increased, renal capsule clearance slightly widened, proximal tubules slightly swollen, small 綆′箣闂撮棿闅欏彉紿，

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