本文選題：自噬 + 氯喹��； 參考：《廣州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：【研究背景】腎結(jié)石是在我國泌尿外科是一種常見病及多發(fā)病,最近一項覆蓋全國的大規(guī)模的橫斷面流行病學(xué)研究表明中國成年人腎結(jié)石患病率為5.8%,結(jié)合我國中國巨大的人口基數(shù),目前中國約有6120萬成年人罹患腎結(jié)石。該研究同時也表明在我國結(jié)石主要集中在華南及西南地區(qū),這與南方的平均氣溫比北方高有關(guān)。腎結(jié)石同時也是全球性疾病,國外多項研究表明在約有5-15%的居民會罹患腎結(jié)石。在所有腎結(jié)石患者中,大約80%腎結(jié)石成分為含鈣結(jié)石,其中草酸鈣結(jié)石是最常見的腎結(jié)石類型,所占比例約為70%-80%。隨著最近幾年腔內(nèi)微創(chuàng)技術(shù)在泌尿系結(jié)石治療方面取得巨大成功,腎結(jié)石患者也因此受益匪淺,但是草酸鈣結(jié)石的發(fā)病因素包括遺傳、環(huán)境、飲食、疾病和代謝等多個方面,其確切病因仍不清楚并且缺乏有效的預(yù)防措施,其在治療后復(fù)發(fā)率仍然很高,大多數(shù)患者一生中因為腎結(jié)石復(fù)發(fā)需要接受多次治療,嚴(yán)重影響患者及其家庭的生活質(zhì)量,同時也給個人及社會都帶來巨大的經(jīng)濟(jì)負(fù)擔(dān)。因此,進(jìn)一步揭示草酸鈣結(jié)石的確切發(fā)病機(jī)制,尋找新的有效干預(yù)靶點,具有重要的科研和臨床價值�！狙芯磕康摹垦芯孔允蓪Υ笫竽I臟草酸鈣晶體形成的影響及可能的作用機(jī)制【研究方法】取健康雄性SD大鼠40只,體重約為200±6.3g,隨機(jī)分為四組,每組10只,分別為對照組、造模組、氯喹干預(yù)組、雷帕霉素干預(yù)組。大鼠腎結(jié)石模型建立方法:自由飲用1%乙二醇28天+1%氯化銨灌胃(2ml/天/只)6天。氯喹干預(yù)組采用氯喹溶液(40mg/kg/day)腹腔注射,雷帕霉素干預(yù)組采用雷帕霉素溶液(0.5mg/kg/day)腹腔注射。連續(xù)飼養(yǎng)28天后,收集大鼠24小時尿液并分析其成石危險相關(guān)成分的含量。取大鼠雙側(cè)腎臟,應(yīng)用免疫組化及透射電鏡方法檢測各組大鼠腎臟自噬水平,并且通過透射電鏡觀察線粒體形態(tài)。通過免疫組化方法檢測各組大鼠腎臟內(nèi)氧化應(yīng)激損傷相關(guān)標(biāo)志物�！狙芯拷Y(jié)果】1.各組大鼠尿液24小時尿危險成分分析與對照組相比,模型組大鼠尿草酸排泄量明顯升高(P0.05),而尿枸櫞酸排泄量明顯降低(P0.05)。與對照組相比,氯喹干預(yù)組大鼠尿PH值及尿鉀、尿氯、尿鈉、尿尿酸、尿枸櫞酸排泄量均明顯降低(P0.05)。與對照組相比,雷帕霉素組大鼠尿氯、尿鉀、尿鈉、尿磷、尿尿酸、尿枸櫞酸排泄量明顯降低(P0.05)。與模型組相比,氯喹干預(yù)組大鼠尿PH值及尿鉀、尿氯、尿鈉、尿磷、尿草酸排泄量明顯降低(P0.05)。與模型組相比,雷帕霉素組大鼠尿PH值及尿氯、尿鉀、尿鈉、尿磷、尿尿酸、尿枸櫞酸排泄量明顯降低(P0.05)。與氯喹干預(yù)組相比,雷帕霉素組大鼠尿尿酸排泄量明顯降低(P0.05),其余尿液指標(biāo)無明顯差異(P0.05)。2.各組大鼠腎臟草酸鈣晶體形成情況對照組大鼠腎臟無可見草酸鈣晶體,而造模組大鼠腎小管內(nèi)可見大量折光性顯著的草酸鈣晶體。與模型組(32.4±5.1/HP)相比,氯喹干預(yù)組(4.0±0.9/HP)大鼠腎臟草酸鈣晶體顯著減少(P0.05),而雷帕霉素干預(yù)組(102.4±8.5/HP)大鼠腎臟草酸鈣晶體則明顯增多(P0.05)。3.透射電鏡下各組大鼠腎臟細(xì)胞自噬水平結(jié)果對照組大鼠腎臟內(nèi)可見單個自噬小體,而模型組大鼠腎臟細(xì)胞內(nèi)可見多量典型的圓球形單層膜自噬溶酶體,其數(shù)量比對照組明顯增多。氯喹干預(yù)組大鼠腎臟細(xì)胞內(nèi)可見單個典型的圓球形單層膜自噬溶酶體。雷帕霉素干預(yù)組大鼠腎臟細(xì)胞內(nèi)可見多量自噬溶酶體。4.免疫組化檢測各組大鼠腎臟內(nèi)自噬標(biāo)志物水平與對照組相比,模型組大鼠腎臟自噬標(biāo)志物L(fēng)C3表達(dá)明顯升高,而自噬標(biāo)志物P62則明顯降低。與模型組相比,氯喹干預(yù)組大鼠腎臟LC3及P62的表達(dá)均明顯升高。與模型組相比,雷帕霉素干預(yù)組大鼠腎臟LC3表達(dá)明顯升高,而P62的表達(dá)明顯降低。5.透射電鏡下各組大鼠腎臟細(xì)胞內(nèi)線粒體形態(tài)觀察對照組大鼠腎臟細(xì)胞內(nèi)可見雙層膜桿狀的線粒體,線粒體內(nèi)、外膜均保持完整,線粒體嵴清晰。模型組大鼠腎臟細(xì)胞內(nèi)線粒體出現(xiàn)損傷,表現(xiàn)為線粒體腫脹,線粒體雙層膜結(jié)構(gòu)模糊,線粒體嵴數(shù)目減少,排列紊亂。氯喹干預(yù)組大鼠腎臟細(xì)胞內(nèi)線粒體結(jié)構(gòu)清晰,與對照組大鼠腎臟細(xì)胞內(nèi)線粒體形態(tài)相似。雷帕霉素干預(yù)組大鼠腎臟細(xì)胞內(nèi)線粒體明顯水腫,線粒體雙層膜結(jié)構(gòu)模糊,線粒體嵴數(shù)目減少,排列紊亂。6.免疫組化檢測各組大鼠腎臟內(nèi)氧化應(yīng)激相關(guān)標(biāo)志物的表達(dá)水平與對照組相比,模型組腎臟的超氧化物歧化酶(SOD)表達(dá)明顯降低,與對照組相比,而單核細(xì)胞趨化蛋白1(MCP-1)與8-羥化脫氧鳥苷(8-OHd G)的表達(dá)則明顯升高。與模型組相比,氯喹干預(yù)組大鼠腎臟的SOD表達(dá)明顯升高,而MCP-1與8-OHd G的表達(dá)則明顯降低。與對照組相比,雷帕霉素干預(yù)組大鼠腎臟的SOD表達(dá)明顯降低,而MCP-1與8-OHd G表達(dá)則明顯升高�！狙芯拷Y(jié)論】1.乙二醇誘導(dǎo)的高草酸尿可明顯提高大鼠腎臟內(nèi)的自噬水平。2.本研究首次證實了自噬在大鼠草酸鈣晶體形成中具有重要的調(diào)控作用,降低大鼠腎臟自噬水平可明顯抑制草酸鈣晶體的形成。3.自噬參與草酸鈣結(jié)石形成的調(diào)控機(jī)制可能是,降低自噬水平抑制了高草酸尿誘導(dǎo)的腎小管上皮細(xì)胞氧化應(yīng)激損傷、線粒體損傷、以及草酸的排泄量,從而抑制了草酸鈣晶體的形成。
[Abstract]:[background] renal calculi is a common disease and frequently occurring disease in the Department of Urology in China. A large-scale cross-sectional epidemiological study covering the country recently shows that the prevalence rate of renal calculi in Chinese adults is 5.8%. In combination with China's huge population base in China, about 61 million 200 thousand adults have kidney stones. It is also indicated that stones in our country are mainly concentrated in Southern China and southwest China, which is related to the higher average temperature in the South than in the north. Kidney stones are also global diseases. A number of studies abroad show that about 5-15% of residents will suffer from kidney stones. In all patients with kidney stones, about 80% of the kidney stones are calcium containing stones, of which calcium oxalate is found. Stone is the most common type of renal calculi, with a proportion of about 70%-80%. with a great success in the treatment of urinary calculi with minimally invasive techniques in recent years. Patients with renal calculi have benefited a lot, but the factors of calcium oxalate stones include heredity, environment, diet, disease and metabolism, and the exact cause is still not. There is a clear and lack of effective preventive measures. The recurrence rate is still high after treatment. Most patients need multiple treatment for the recurrence of kidney stones in their life, which seriously affects the quality of life of the patients and their families, and also brings huge economic burden to both individuals and society. Therefore, the exact results of calcium oxalate stones are further revealed. The study aims to study the effect of autophagy on the formation of calcium oxalate crystals in rats and its possible mechanism of action. [research methods] 40 healthy male SD rats, with a weight of about 200 6.3g, were randomly divided into four groups, 10 in each group, and the control group, respectively. Model establishment, chloroquine intervention group, rapamycin intervention group. Rats kidney stone model establishment method: free drinking 1% glycol 28 days +1% ammonium chloride gavage (2ml/ days / only) 6 days. Chloroquine intervention group with chloroquine solution (40mg/kg/day) intraperitoneal injection, rapamycin intervention group using reparamycin solution (0.5mg/kg/day) intraperitoneal injection. Continuous feeding for 28 days, The urine of 24 hours of rats was collected and the content of the related components of the risk of the stone formation was analyzed. The renal autophagy was detected by immunohistochemistry and transmission electron microscopy, and the morphology of the mitochondria was observed by transmission electron microscopy. The related markers of oxidative stress in the kidneys of the rats were detected by immunohistochemistry. [results] 1. compared with the control group, the urine oxalic acid excretion of rats in the model group was significantly increased (P0.05) and the excretion of urinary citric acid decreased significantly (P0.05). Compared with the control group, the urine pH value, urinary potassium, urinary chlorine, urine sodium, ururic acid and urinary citric acid excretion were significantly higher in the chloroquine intervention group. Lower (P0.05). Compared with the control group, the urine chlorine, urine potassium, urine sodium, urine phosphorus, ururic acid and urinary citric acid excretion decreased significantly (P0.05) in the control group. Compared with the model group, the urine pH and urinary potassium, urine chlorine, urine sodium, urine phosphorus and urine oxalic acid discharge were significantly reduced (P0.05). Compared with the model group, the urine pH value of the rat group was compared with the model group. Urinary potassium, urine potassium, urine sodium, urine phosphorus, urination acid, urinary citric acid excretion decreased significantly (P0.05). Compared with the chloroquine intervention group, the urinary excretion of ureic acid in the rapamycin group was significantly lower (P0.05), and the other urine indexes were not significantly different (P0.05) in the kidney of.2. rats, the renal calcium oxalate crystals were not visible in the control group. A large number of refracted calcium oxalate crystals were visible in the renal tubules of the model rats. Compared with the model group (32.4 5.1/HP), the renal calcium oxalate crystals in the rats in the chloroquine intervention group (4 0.9/HP) were significantly reduced (P0.05), while the renal calcium oxalate crystals in the rapamycin group (102.4 + 8.5/HP) were significantly increased (P0.05).3. transmission electron microscopy The autophagy level of the rat kidney cells showed a single autophagosome in the kidney of the control group, while a number of typical spherical monolayer autophagic lysosomes were found in the rat kidney cells in the model group. The number of the autophagic lysosomes in the renal cells of the rats was significantly higher than that in the control group. Compared with the control group, the expression of autophagic markers in kidneys of rats in the renal cells of the renal cells of rapamycin intervention group was significantly higher than that in the control group. The autophagic marker P62 was significantly increased in the model group, while the autophagic marker P62 was significantly reduced in the model group. Compared with the model group, the renal LC3 and P of the rats in the chloroquine intervention group were LC3 and P. The expression of 62 was significantly increased. Compared with the model group, the expression of LC3 in the kidneys of the rats with rapamycin intervention was significantly increased, while the expression of P62 significantly decreased the mitochondrial morphology in the renal cells of the rats under.5. transmission electron microscopy. The mitochondrial crista was clear. The mitochondria in the renal cells of the rat model group were damaged in the model group, showing the swelling of mitochondria, the blurred structure of the mitochondrial double layer membrane, the decrease of the mitochondrial crista and the disorder. The mitochondria structure of the renal cells in the renal cells of the rats in the chloroquine intervention group was similar to the mitochondria in the kidney of the control group. The mitochondria of the renal cells were edema, the structure of the mitochondrial double layer membrane was blurred and the number of mitochondrial crista decreased. The expression level of the oxidative stress related markers in the kidneys of each group was compared with the control group. Compared with the control group, the expression of superoxide dismutase (SOD) in the model group was significantly lower than that of the control group, but compared with the control group, the single nucleus was compared with the control group. The expression of cell chemoattractant protein 1 (MCP-1) and 8- hydroxylated deoxy guanosine (8-OHd G) increased significantly. Compared with the model group, the expression of SOD in the kidneys of the rats in the chloroquine intervention group was significantly increased, while the expression of MCP-1 and 8-OHd G decreased significantly. Compared with the control group, the expression of SOD in the kidneys of the rapamycin group was significantly reduced, while MCP-1 and 8-OHd G were expressed. [study conclusion] 1. ethylene glycol induced high oxalate urine can obviously improve the autophagy level in the kidney of the rat.2.. This study was the first to confirm that autophagy plays an important role in the formation of calcium oxalate crystals in rats. Reducing the level of autophagy in the rat kidney can obviously inhibit the formation of calcium oxalate in.3. autophagy. The regulation mechanism of the formation of stones may be that the reduction of autophagy inhibits the oxidative stress of renal tubular epithelial cells induced by high oxalate, mitochondrial damage, and the excretion of oxalic acid, which inhibits the formation of calcium oxalate crystals.

【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R692.4

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