自噬对大鼠肾脏草酸钙晶体形成的影响及作用机制研究

发布时间：2018-05-03 22:02

本文选题：自噬 + 氯喹　；参考：《广州医科大学》2017年硕士论文

【摘要】：【研究背景】肾结石是在我国泌尿外科是一种常见病及多发病,最近一项覆盖全国的大规模的横断面流行病学研究表明中国成年人肾结石患病率为5.8%,结合我国中国巨大的人口基数,目前中国约有6120万成年人罹患肾结石。该研究同时也表明在我国结石主要集中在华南及西南地区,这与南方的平均气温比北方高有关。肾结石同时也是全球性疾病,国外多项研究表明在约有5-15%的居民会罹患肾结石。在所有肾结石患者中,大约80%肾结石成分为含钙结石,其中草酸钙结石是最常见的肾结石类型,所占比例约为70%-80%。随着最近几年腔内微创技术在泌尿系结石治疗方面取得巨大成功,肾结石患者也因此受益匪浅,但是草酸钙结石的发病因素包括遗传、环境、饮食、疾病和代谢等多个方面,其确切病因仍不清楚并且缺乏有效的预防措施,其在治疗后复发率仍然很高,大多数患者一生中因为肾结石复发需要接受多次治疗,严重影响患者及其家庭的生活质量,同时也给个人及社会都带来巨大的经济负担。因此,进一步揭示草酸钙结石的确切发病机制,寻找新的有效干预靶点,具有重要的科研和临床价值。【研究目的】研究自噬对大鼠肾脏草酸钙晶体形成的影响及可能的作用机制【研究方法】取健康雄性SD大鼠40只,体重约为200±6.3g,随机分为四组,每组10只,分别为对照组、造模组、氯喹干预组、雷帕霉素干预组。大鼠肾结石模型建立方法:自由饮用1%乙二醇28天+1%氯化铵灌胃(2ml/天/只)6天。氯喹干预组采用氯喹溶液(40mg/kg/day)腹腔注射,雷帕霉素干预组采用雷帕霉素溶液(0.5mg/kg/day)腹腔注射。连续饲养28天后,收集大鼠24小时尿液并分析其成石危险相关成分的含量。取大鼠双侧肾脏,应用免疫组化及透射电镜方法检测各组大鼠肾脏自噬水平,并且通过透射电镜观察线粒体形态。通过免疫组化方法检测各组大鼠肾脏内氧化应激损伤相关标志物。【研究结果】1.各组大鼠尿液24小时尿危险成分分析与对照组相比,模型组大鼠尿草酸排泄量明显升高(P0.05),而尿枸橼酸排泄量明显降低(P0.05)。与对照组相比,氯喹干预组大鼠尿PH值及尿钾、尿氯、尿钠、尿尿酸、尿枸橼酸排泄量均明显降低(P0.05)。与对照组相比,雷帕霉素组大鼠尿氯、尿钾、尿钠、尿磷、尿尿酸、尿枸橼酸排泄量明显降低(P0.05)。与模型组相比,氯喹干预组大鼠尿PH值及尿钾、尿氯、尿钠、尿磷、尿草酸排泄量明显降低(P0.05)。与模型组相比,雷帕霉素组大鼠尿PH值及尿氯、尿钾、尿钠、尿磷、尿尿酸、尿枸橼酸排泄量明显降低(P0.05)。与氯喹干预组相比,雷帕霉素组大鼠尿尿酸排泄量明显降低(P0.05),其余尿液指标无明显差异(P0.05)。2.各组大鼠肾脏草酸钙晶体形成情况对照组大鼠肾脏无可见草酸钙晶体,而造模组大鼠肾小管内可见大量折光性显著的草酸钙晶体。与模型组(32.4±5.1/HP)相比,氯喹干预组(4.0±0.9/HP)大鼠肾脏草酸钙晶体显著减少(P0.05),而雷帕霉素干预组(102.4±8.5/HP)大鼠肾脏草酸钙晶体则明显增多(P0.05)。3.透射电镜下各组大鼠肾脏细胞自噬水平结果对照组大鼠肾脏内可见单个自噬小体,而模型组大鼠肾脏细胞内可见多量典型的圆球形单层膜自噬溶酶体,其数量比对照组明显增多。氯喹干预组大鼠肾脏细胞内可见单个典型的圆球形单层膜自噬溶酶体。雷帕霉素干预组大鼠肾脏细胞内可见多量自噬溶酶体。4.免疫组化检测各组大鼠肾脏内自噬标志物水平与对照组相比,模型组大鼠肾脏自噬标志物LC3表达明显升高,而自噬标志物P62则明显降低。与模型组相比,氯喹干预组大鼠肾脏LC3及P62的表达均明显升高。与模型组相比,雷帕霉素干预组大鼠肾脏LC3表达明显升高,而P62的表达明显降低。5.透射电镜下各组大鼠肾脏细胞内线粒体形态观察对照组大鼠肾脏细胞内可见双层膜杆状的线粒体,线粒体内、外膜均保持完整,线粒体嵴清晰。模型组大鼠肾脏细胞内线粒体出现损伤,表现为线粒体肿胀,线粒体双层膜结构模糊,线粒体嵴数目减少,排列紊乱。氯喹干预组大鼠肾脏细胞内线粒体结构清晰,与对照组大鼠肾脏细胞内线粒体形态相似。雷帕霉素干预组大鼠肾脏细胞内线粒体明显水肿,线粒体双层膜结构模糊,线粒体嵴数目减少,排列紊乱。6.免疫组化检测各组大鼠肾脏内氧化应激相关标志物的表达水平与对照组相比,模型组肾脏的超氧化物歧化酶(SOD)表达明显降低,与对照组相比,而单核细胞趋化蛋白1(MCP-1)与8-羟化脱氧鸟苷(8-OHd G)的表达则明显升高。与模型组相比,氯喹干预组大鼠肾脏的SOD表达明显升高,而MCP-1与8-OHd G的表达则明显降低。与对照组相比,雷帕霉素干预组大鼠肾脏的SOD表达明显降低,而MCP-1与8-OHd G表达则明显升高。【研究结论】1.乙二醇诱导的高草酸尿可明显提高大鼠肾脏内的自噬水平。2.本研究首次证实了自噬在大鼠草酸钙晶体形成中具有重要的调控作用,降低大鼠肾脏自噬水平可明显抑制草酸钙晶体的形成。3.自噬参与草酸钙结石形成的调控机制可能是,降低自噬水平抑制了高草酸尿诱导的肾小管上皮细胞氧化应激损伤、线粒体损伤、以及草酸的排泄量,从而抑制了草酸钙晶体的形成。
[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.

【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692.4

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