本文選題：橫紋肌溶解 + 急性腎損傷��； 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:橫紋肌溶解(rhabdomyolysis,RM)所致急性腎損傷(acute kidney injury,AKI)為腎臟常見病,如不及時處理可發(fā)展為不可逆腎臟損害。擠壓傷、過度訓(xùn)練、外傷、中毒等理化因素可以引起橫紋肌溶解,進而導(dǎo)致急性腎損傷,但其確切機制尚不完全清楚。因此,探究其發(fā)病機制,進行早期干預(yù)治療,以減輕腎臟損害,從而改善預(yù)后,降低死亡率,已成為軍事醫(yī)學(xué)、運動醫(yī)學(xué)及臨床醫(yī)學(xué)的重要課題。本研究旨在通過甘油肌注建立大鼠橫紋肌溶解致急性腎損傷模型,擬評價RM時大鼠腎組織Sonic Hedgehog(Shh)信號轉(zhuǎn)導(dǎo)通路中關(guān)鍵蛋白的表達變化及其意義,觀察抗膽堿藥對其表達變化的影響,為臨床研究提供參考。方法:1實驗分組:將體重在200-220g之間的72只SD雄性大鼠用隨機數(shù)字表法平均分為4組。對照組(Control,CN,n=18)、急性腎損傷組(acute kidney injury,AKI,n=18)、山莨菪堿干預(yù)組(anisodmine,AD,n=18)、鹽酸戊乙奎醚干預(yù)組(penehyclidine hydrochloride,PHC,n=18),根據(jù)觀察時間又分為3個亞組,即注射后6h、24h、72h,每個時間點6只大鼠。2模型制備:將大鼠禁食水,CN組18只大鼠于雙后腿內(nèi)側(cè)注射生理鹽水,用量10ml/kg。AKI組、AD組、和PHC組雙側(cè)后肢肌肉注射50%甘油生理鹽水10 ml/kg,建立大鼠急性腎損傷模型。AD組18只大鼠在注射甘油前的20min,于腹腔注射山莨菪堿,用量10 mg/kg,PHC組18只大鼠在甘油注射前20min,于腹腔注射鹽酸戊乙奎醚,用量2mg/kg。隨后各組大鼠均給予常規(guī)飼料自由進食、水、觀察大鼠一般情況。3取材處理:各組于相應(yīng)時間點分別隨機取6只大鼠,于腹腔注入戊巴比妥(60 mg/kg),深度麻醉后大鼠腹部正中切開,于上腔靜脈抽取靜脈血樣,生化分析法測定血漿尿素氮(BUN)、肌酐(Cr)、比色法測血漿、腎髓質(zhì)髓過氧化物酶(MPO)。取雙腎,去除表面脂肪筋膜,右腎放置在-80℃冰箱凍存,采用Western blot法檢測Shh、Gli2蛋白的表達;左腎縱行剖開,10%中性福爾馬林固定,常規(guī)脫水,石蠟包埋,切片;應(yīng)用HE染色光學(xué)顯微鏡下觀察腎組織結(jié)構(gòu)變化,并行腎小管損傷評分,評估損傷程度;采用免疫組化染色(PV法)觀察腎組織Shh、Gli2的表達情況。4統(tǒng)計分析:采用SPSS 21.0軟件進行統(tǒng)計學(xué)分析,以均數(shù)±標準差(x±s)表示。各組間比較采用單因素方差分析(one-way ANOVA)分析,兩兩比較采用Student-Newman-Kuels t檢驗,以P0.05為有統(tǒng)計學(xué)意義。結(jié)果:1各組血BUN、Cr變化:與對照組各時間點比較,急性腎損傷組大鼠血清Cr和BUN均增高(P0.05),且進行性升高。山莨菪堿干預(yù)組、鹽酸戊乙奎醚干預(yù)組血清Cr、BUN在24h、72h較AKI組同時間點明顯降低(P0.05),但仍高于對照組(P0.05),其中AD組24h、72h血清Cr、BUN較同期PHC組高;2各組血、腎組織MPO變化:與對照組比較,AKI組6小時組MPO明顯升高(P0.05),24小時達到高峰、72小時組血MPO下降,但仍高于對照組(P0.05)。與AKI組比較,山莨菪堿干預(yù)組、鹽酸戊乙奎醚干預(yù)組各時間點MPO降低(P0.05),但仍高于對照組(P0.05),與AD組同期比較,PHC血MPO6h、24h,腎MPO24h、72h較低;3各組腎組織病理變化:對照組大鼠雙腎病理形態(tài)學(xué)未見明顯改變。急性腎損傷組肌注甘油后6小時可見腎組織充血腫脹,腎小管上皮細胞腫脹、變性,少量壞死、脫落。管腔中形成蛋白管型,腎間質(zhì)少量炎性細胞浸潤。24小時組腎小管上皮細胞變性、壞死加重,大量蛋白管型,間質(zhì)可見大量炎性細胞浸潤,72小時組腎小管管腔內(nèi)管型逐漸溶解消失,偶可見間斷性損傷部位的再生上皮細胞,炎癥減輕。山莨菪堿干預(yù)組、鹽酸戊乙奎醚干預(yù)組各時間點亞組上皮細胞變性、壞死程度較急性腎損傷組減輕,腎小管再生增加,腎小管損傷評分,與CN組比較,AKI組6h、24h、72h腎小管損傷評分逐漸升高(P0.05),AD、PHC組腎小管損傷評分較同期AKI組低,但高于CN組(P0.05),與AD組比較,PHC6h組評分較低(P0.05);4各組腎組織Gli2及Shh表達的變化:免疫組化顯示,Gli2在正常對照組的腎小管上皮細胞中呈弱表達,急性腎損傷組大鼠于6小時組腎組織Gli2表達增強(P0.05),24小時表達量增加(P0.05),72小時進一步增強(P0.05)。山莨菪堿組、鹽酸戊乙奎醚組各時間點蛋白表達均高于急性腎損傷組(P0.05)。Shh在對照組大鼠腎小管上皮細胞表達。急性腎損傷組Shh于甘油肌注后6小時表達減弱(P0.05),24小時組Shh組表達增強(P0.05)。72小時組,Shh表達持續(xù)升高(P0.05)。山莨菪堿組、鹽酸戊乙奎醚組各時間點與急性腎損傷組同期比較表達增強(P0.05),與AD組同期比較,PHC組Shh 6h、24h的表達,Gli2 24h、72h的表達較強(P0.05)。Western blot結(jié)果顯示與免疫組化結(jié)果一致。結(jié)論:1橫紋肌溶解致急性腎損傷時,腎組織Shh蛋白早期表達減弱,后期表達升高,說明橫紋肌溶解致急性腎損傷可再度激活Shh信號通路。2 Shh信號通路中關(guān)鍵蛋白Shh、Gli2在24小時、72小時表達上調(diào),表明激活Shh信號通路可能對急性腎損傷腎組織保護、修復(fù)發(fā)揮重要作用。3抗膽堿藥山莨菪堿、鹽酸戊乙奎醚可能部分通過激活Shh信號通路,減輕急性腎損傷,促進損傷組織早期修復(fù),為臨床用藥提供參考。
[Abstract]:Objective: acute kidney injury (AKI) is a common kidney disease (acute kidney injury, AKI) caused by rhabdomyolysis (acute). If untimely treatment can be developed into an irreversible renal damage, the physical and chemical factors such as extrusion injury, overtraining, trauma and poisoning can cause rhabdomyolysis and lead to acute renal injury, but the exact mechanism is not completely clear. Therefore, it has become an important subject in military medicine, sports medicine and clinical medicine to explore the pathogenesis and early intervention treatment in order to reduce the renal damage, improve the prognosis and reduce the mortality. This study aims to establish a rat model of acute renal injury induced by rhabdomyolysis through the injection of glycerol muscle, and to evaluate the renal tissue Sonic in RM rats. The changes in the expression of key proteins in the Hedgehog (Shh) signal transduction pathway and its significance were observed to provide reference for clinical study. Methods: 1 experimental groups were divided into 4 groups of 72 SD male rats with weight between 200-220g and the control group (Control, CN, n=18), and acute renal injury. Group (acute kidney injury, AKI, n=18), anisodamine intervention group (anisodmine, AD, n=18), amyl hydrochloric ether intervention group (penehyclidine hydrochloride, PHC, n=18), according to the observation time, divided into 3 subgroups, namely, after the injection, 6 rats were prepared at each time point: the rat fasting water, 18 rats in the inside of the double hind legs In group 10ml/kg.AKI, group AD, group AD, and group PHC, the muscles of bilateral hind limbs were injected with 50% glycerol 10 ml/kg, and 18 rats in group.AD were injected with Anisodamine before glycerol injection, and the dosage of anisodamine was injected into the abdominal cavity, and 18 rats in group PHC were injected with glutamine hydrochloride before glycerol injection. The rats were given free feeding of conventional feed after the dosage of 2mg/kg., and water was given to the rats. The rats were treated with.3. 6 rats were randomly selected at the corresponding time points and injected into the abdominal cavity with pentobarbital (60 mg/kg). After deep anesthesia, the abdominal median incision was taken, the venous blood samples were extracted from the superior vena cava and the biochemical analysis was used to determine the blood. Plasma urea nitrogen (BUN), creatinine (Cr), colorimetric method to measure plasma and renal medullary myeloperoxidase (MPO), take two kidneys, remove the surface of the fat fascia, the right kidney is placed at -80 centigrade refrigerator and frozen, the expression of Shh, Gli2 protein is detected by Western blot; the left kidney is opened in a longitudinal way, 10% neutral formalin is fixed, routine dehydration, paraffin embedding and slicing; HE staining light is applied. The changes of renal tissue structure were observed under the microscope, the renal tubule injury score was evaluated and the degree of injury was evaluated. The expression of Shh and Gli2 in renal tissue was observed by immunohistochemical staining (PV method).4 statistical analysis: SPSS 21 software was used for statistical analysis with mean standard deviation (x + s). The single factor variance analysis (one-wa) was used in each group. Y ANOVA) analysis, 22 compared with Student-Newman-Kuels t test, with P0.05 as a statistical significance. Results: 1 groups of blood BUN, Cr changes: compared with the control group, the serum Cr and BUN increased in acute renal injury rats (P0.05), and progressively increased. Compared with the AKI group at the same time point (P0.05), it was still higher than the control group (P0.05), and the serum Cr and BUN in AD group were higher than that of the same group in the same period, and BUN was higher than that of the same group in the same period. 2 groups of blood and renal tissue MPO changes: compared with the control group, the MPO significantly increased in the 6 hour group of AKI group, reached the peak at 24 hours, and decreased in the 72 hour group, but still higher than the control group. In the group of anisodamine intervention, MPO decreased (P0.05) at all time points in the treatment group, but it was still higher than the control group (P0.05). Compared with the AD group, PHC blood MPO6h, 24h, renal MPO24h and 72h were lower. 3 the pathological changes of kidney tissues in the control group were not obviously changed. The kidney was seen in the acute renal injury group after 6 hours of glycerol. Tissue hyperemia and swelling, renal tubular epithelial cells swelling, degeneration, a small amount of necrosis, shedding. The tubule formation in the lumen, a small amount of inflammatory cells in the renal interstitial cells infiltrated.24 hours, the renal tubular epithelial cells degeneration, necrosis aggravated, a large number of protein tube type, mass of inflammatory cells infiltration, 72 hours group of renal tubules gradually dissolved in the group. I can see the regenerative epithelial cells in the discontinuous injury site and the inflammation alleviated. In the anisodamine intervention group, the epithelial cell degeneration in each time point group of the amyl hydrochloric acid group, the necrosis degree was less than the acute renal injury group, the renal tubule regeneration increased and the renal tubule injury score was increased. Compared with the CN group, the 6h, 24h and 72h renal tubule injury scores in group AKI increased gradually. (P0.05), the score of renal tubule injury in group AD and PHC was lower than that of group AKI, but higher than that in group CN (P0.05). Compared with group AD, the score of PHC6h group was lower (P0.05); 4 the changes of Gli2 and Shh expression in renal tissue in each group: immunohistochemistry showed that Gli2 was weakly expressed in the tubule epithelial cells of the normal control group, and the rats in the acute renal injury group were in the 6 hour group of renal tissue. Enhancement (P0.05), increased expression of 24 hours (P0.05) and further enhancement (P0.05). The expression of each time point in the group of anisodamine and amyl hydrochloric acid was higher than that in the acute renal injury group (P0.05).Shh in the renal tubular epithelial cells in the control group. The expression of Shh in the acute renal injury group was weakened at 6 hours after the glycerol injection (P0.05) and 24 hours. Group Shh expression enhanced (P0.05).72 hour group and Shh expression increased continuously (P0.05). The time points of anisodamine group and amyl hydrochloric acid group were compared with those of acute renal injury group at the same time (P0.05). The expression of Shh 6h, 24h expression in group PHC was compared with that of group AD in the same period of the AD group. Conclusion: when 1 rhabdomyolysis induced acute renal injury, the early expression of Shh protein in renal tissue decreased and later expression increased, indicating that rhabdomyolysis induced acute renal injury could reactivate the key protein Shh in the Shh signaling pathway.2 Shh signaling pathway, Gli2 at 24 hours, up regulation up to 72 hours, indicating that activation of Shh signaling pathway may be associated with acute renal damage. Renal tissue protection and repair play an important role in.3 anticholine drug anisodamine. Amyl hydrochloric acid may partially activate Shh signaling pathway to alleviate acute renal injury and promote early repair of damaged tissue, and provide reference for clinical use.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R692;R685.5

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