泛素连接酶Parkin在糖尿病肾病肾小管上皮细胞应激性衰老中的作用

发布时间：2018-04-30 15:12

本文选题：糖尿病肾病 + 应激性衰老　；参考：《第三军医大学》2017年硕士论文

【摘要】：糖尿病肾病(Diabetic Nephropathy,DN)是糖尿病的主要并发症之一,是目前世界范围内导致终末期肾脏病及透析的首要病因。DN发病机制复杂,其中肾小管上皮细胞应激性衰老是导致DN发生发展的重要机制。研究证实,肾小管细胞衰老是DN病理损伤标志之一。衰老肾小管细胞能够产生大量促炎因子和促纤维化因子等衰老相关分泌表型,引起组织局部炎症反应和纤维化。同时抑制肾小管的加速衰老可延缓DN进展为终末期肾脏疾病。E3泛素连接酶Parkin在脑、心、肾等组织器官中均可广泛表达,其基因突变与老年性痴呆、帕金森病的发病密切相关。过表达Parkin能够延长果蝇寿限,提示Parkin可能是一个潜在的抗衰老因子,但目前Parkin与DN肾脏衰老关系尚不清楚。本研究旨在探讨Parkin在DN诱导的肾小管细胞应激性衰老中的作用,为DN的防治寻找新的有效靶点和理论依据。一、实验方法1.临床病例研究对象为2015年1月至2016年6月期间于我科就诊的2型糖尿病患者。行肾脏病理确诊为DN共40例。对照组来源于肾脏原发性肿瘤患者行切除术后远端正常肾组织,并行肾组织病理检查,共10例。收集两组基线临床数据,包括年龄、性别、身高、体重、体质指数、收缩压、舒张压。于肾活检或手术1天前收集患者血尿标本,-80℃超低温保存,测定其生化指标包括:血肌酐、血清尿素氮、血糖、糖化血红蛋白、血清胱抑C、血清白蛋白、血尿酸、血清总胆固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白、C反应蛋白、尿蛋白定量、尿N-乙酰-β-氨基葡萄糖苷酶和估算肾小球滤过率。免疫组化检测糖尿病肾病患者肾组织Parkin的表达,显微镜下观察并计算Parkin阳性率,并与临床资料和病理损伤评分进行相关性分析。免疫荧光检测糖尿病肾病患者肾组织Parkin分别与衰老标志物p16、衰老相关分泌表型IL-6和TGF-β的关系。2.体外实验剪取3-4周龄的C57雄性鼠双肾皮质,经胶原酶孵育及双重筛网滤过后获得肾小管细胞。采用原代肾小管细胞专用培养基,常规培养两代后用于后续实验。通过30mmol/l高糖刺激肾小管细胞72h,并通过免疫荧光检测肾小管细胞p16、γ-H2AX的阳性率,WB检测p16和Parkin蛋白水平的变化,PCR检测Parkin mRNA水平的变化,ELISA检测细胞培养基上清中IL-6和TGF-β的分泌。进一步采用Parkin过表达腺病毒或Parkin siRNA转染肾小管细胞,检测Parkin上调或下调后高糖刺激的肾小管细胞p16、γ-H2AX的表达变化,及IL-6和TGF-β的分泌。二、结果1.糖尿病肾病肾组织Parkin的表达变化及与肾小管细胞应激性衰老、肾间质损伤的关系免疫组化显示,Parkin主要表达于肾小管上皮细胞。对照组肾小管细胞Parkin阳性率为43.5±6.0%,IFTA 0分组肾小管细胞Parkin阳性率与对照组相近,为40.8±4.3%,IFTA 1分组为32.8±4.8%,IFTA 2分组为28.5±4.2%,IFTA 3分组为19.0±4.9%,提示肾小管细胞Parkin的阳性率随着DN的进展逐渐降低。相关性分析显示Parkin的阳性率与肾小球硬化、肾小管萎缩与肾间质纤维化、肾间质炎症均呈显著负相关(P0.05),与肾脏功能损伤指标,包括尿蛋白定量、尿NAG、尿素氮、血清胱抑素C、血肌酐和收缩压均呈负相关,与e GFR呈正相关(P0.05)。表明Parkin表达降低与DN肾结构和功能损伤水平均密切相关。进一步通过免疫荧光检测糖尿病肾病患者肾组织肾小管细胞Parkin分别与p16、IL-6及TGF-β的关系发现,与对照组相比,DN患者肾小管细胞衰老标志物p16、促炎性因子IL-6、促纤维化因子TGF-β的表达增多,与Parkin的表达趋势相反。2.Parkin与DN肾小管上皮细胞应激性衰老的体外研究2.1高糖诱导肾小管细胞应激性衰老体外研究发现,与正常对照组相比,高糖组肾小管细胞p16和γ-H2AX的阳性率显著增大(P0.05);WB结果表明高糖组p16蛋白表达水平为对照组的2.13倍(P0.05);ELISA表明肾小管细胞IL-6、TGF-β的分泌显著增多(P0.05),提示高糖环境可诱导肾小管细胞应激性衰老及IL-6、TGF-β的分泌。2.2高糖刺激下肾小管细胞Parkin的表达变化与对照组相比,高糖刺激后肾小管细胞Parkin的mRNA水平下降了60.8%,蛋白水平则下降了53.1%(P0.05),与DN患者中Parkin表达逐渐减少的趋势相同,提示高糖可抑制肾小管细胞Parkin的表达。2.3.Parkin对高糖诱导的肾小管细胞应激性衰老的作用Parkin过表达腺病毒转染上调Parkin的表达后发现,与高糖组比较,Parkin过表达后肾小管细胞p16和γ-H2AX的阳性率分别减少了32.1%和37.8%(P0.05),WB提示该组p16蛋白水平减少了18.4%(P0.05,vs高糖组);Parkin siRNA沉默Parkin发现,高糖刺激后,Parkin干扰组p16和γ-H2AX的阳性肾小管细胞比例较单纯高糖刺激组升高了24.5%和17.6%(P0.05);WB提示该组p16蛋白水平升高了21.0%(P0.05,vs高糖组),表明Parkin可抑制高糖诱导的肾小管细胞应激性衰老。进一步检测细胞培养基上清IL-6和TGF-β的分泌,发现高糖+Parkin siRNA组肾小管细胞IL-6和TGF-β的分泌较高糖组均显著增强,同时高糖+Parkin过表达组IL-6和TGF-β的分泌较高糖组显著减少(P0.05),表明Parkin可抑制高糖诱导的肾小管细胞IL-6、TGF-β的分泌。三、结论E3泛素连接酶Parkin的表达随着DN肾间质损伤的加重而逐渐减少,并与DN肾组织结构与功能损伤、肾小管细胞应激性衰老密切相关;Parkin可抑制高糖诱导的肾小管上皮细胞应激性衰老和促炎促纤维化因子的分泌。
[Abstract]:Diabetic Nephropathy (DN) is one of the major complications of diabetes. It is the leading cause of end-stage renal disease and dialysis in the world. The pathogenesis of.DN is complex, and the stress senescence of renal tubular epithelial cells is an important mechanism for the development of DN. It is confirmed that the aging of renal tubular cells is the pathology of DN. One of the damage markers. Aging renal tubule cells can produce a large number of aging related secretory phenotypes, such as proinflammatory factors and fibrotic factors, and cause local inflammatory response and fibrosis in the tissue. Inhibition of accelerated aging of renal tubules can delay the progression of DN to.E3 ubiquitin linked enzyme Parkin in the end stage renal disease in the brain, heart, kidney and other tissues and organs. It is widely expressed that its gene mutation is closely related to the onset of Alzheimer's disease and Parkinson's disease. Overexpression of Parkin can prolong the life limit of Drosophila, suggesting that Parkin may be a potential antiaging factor, but the relationship between Parkin and DN renal senescence is not yet clear. The purpose of this study was to explore the stress senescence of Parkin induced renal tubular cells in DN. The role of the study was to find new effective targets and theoretical bases for the prevention and control of DN. 1. Experimental methods 1. clinical cases were studied in patients with type 2 diabetes in our department from January 2015 to June 2016. A total of 40 cases of renal pathology were diagnosed as DN, and the control group was derived from the normal renal tissue after resection of the primary renal tumor. Two groups of baseline clinical data, including age, sex, height, body weight, body mass index, systolic blood pressure, and diastolic blood pressure, were collected in 10 cases, including blood creatinine, serum urea nitrogen, blood sugar, glycated hemoglobin, cysteine, and serum cystine. C, serum albumin, blood uric acid, serum total cholesterol, triglycerides, high density lipoprotein, low density lipoprotein, C reactive protein, urine protein quantitative, urinary N- acetyl - beta aminoglucosidase and estimated glomerular filtration rate. Immunohistochemistry was used to detect the expression of Parkin in renal tissue of diabetic nephropathy patients and to observe and calculate Parkin positive under microscope. Correlation analysis of clinical data and pathological damage score. Immunofluorescence detection of renal tissue Parkin in patients with diabetic nephropathy, p16, senescence related secretory phenotype IL-6 and TGF- beta,.2. in vitro experimental clipping of two renal cortex of 3-4 weeks old male C57 male rats, obtained after collagenase incubation and double screen filtration. Renal tubule cells were used for primary renal tubule cell specific culture medium. After two generations of routine culture, the renal tubule cells were used for follow-up experiment. 72h of renal tubule cells was stimulated by 30mmol/l high glucose. The positive rate of renal tubule cells p16, gamma -H2AX was detected by immunofluorescence. The changes of p16 and Parkin protein levels were detected by WB. The change of Parkin mRNA level was detected by PCR, and the ELISA detection was detected. The secretion of IL-6 and TGF- beta in the supernatant of cell culture medium. Further transfection of renal tubule cells with Parkin overexpression adenovirus or Parkin siRNA to detect the expression of p16, the expression of gamma -H2AX, and the secretion of IL-6 and TGF- beta in renal tubule cells stimulated by Parkin up or down. Two, the expression of Parkin in the renal tissue of 1. diabetic nephropathy The relationship between renal tubular cells stress senescence and renal interstitial damage showed that Parkin was mainly expressed in renal tubular epithelial cells. The positive rate of Parkin in renal tubule cells in the control group was 43.5 + 6%. The Parkin positive rate of renal tubular cells in IFTA 0 groups was similar to that of the control group, 40.8 + 4.3%, 32.8 + 4.8% in IFTA 1, and 28.5 + 4.2%, IF in the IFTA 2 group. IF The TA 3 group was 19 + 4.9%, suggesting that the positive rate of Parkin in renal tubule cells gradually decreased with the progression of DN. The correlation analysis showed that the positive rate of Parkin was negatively correlated with glomerulosclerosis, renal tubule atrophy and renal interstitial fibrosis and renal interstitial inflammation (P0.05), and the renal function damage index, including urine protein quantitative, urine NAG, urea nitrogen, Serum cystatin C, serum creatinine and systolic pressure were negatively correlated with e GFR (P0.05). It was indicated that the decrease of Parkin expression was closely related to the level of DN renal structure and function damage. The relationship between Parkin and p16, IL-6 and TGF- beta in renal tubular cells of renal tissue of diabetic nephropathy patients was detected by immunofluorescence. Comparison of DN patients' renal tubular cell senescence marker p16, proinflammatory factor IL-6, and fibrotic factor TGF- beta expression increase, in contrast to Parkin expression trend in vitro,.2.Parkin and DN renal tubular epithelial cells stress senescence in vitro, 2.1 high sugar induced renal tubular cells stress senescence in vitro, compared with the normal control group, high glucose group The positive rate of p16 and gamma -H2AX in renal tubule cells increased significantly (P0.05), and WB results showed that the expression of p16 protein in the high glucose group was 2.13 times as high as that of the control group (P0.05). ELISA showed that the secretion of IL-6 and TGF- beta in renal tubule cells increased significantly (P0.05), suggesting that high glucose environment could induce stress senescence and IL-6 of renal tubule cells, and the secretion of TGF- beta was stimulated by high glucose. Compared with the control group, the expression of Parkin in renal tubule cells decreased by 60.8%, and the protein level decreased by 53.1% (P0.05) after high glucose stimulation, which was the same as the decreasing trend of Parkin expression in DN patients, suggesting that high glucose could inhibit the expression of renal tubule fine cell Parkin in renal tubules induced by high glucose. The effect of Parkin overexpression adenovirus transfection on Parkin expression was found. Compared with high glucose group, the positive rate of p16 and gamma -H2AX in renal tubular cells decreased by 32.1% and 37.8% (P0.05) after Parkin overexpression, WB indicated that the level of p16 protein in this group decreased by 18.4% (P0.05, vs high glucose group); Parkin siRNA was silent. After high glucose stimulation, the proportion of p16 and gamma -H2AX positive tubular cells in Parkin interference group increased by 24.5% and 17.6% (P0.05). WB suggested that the level of p16 protein in this group increased by 21% (P0.05, vs high sugar group), indicating that Parkin could inhibit high glucose induced tubule cell stress senescence. Further detection of cell culture supernatant IL-6. With the secretion of TGF- beta, it was found that the secretion of IL-6 and TGF- beta in the renal tubular cells of the high sugar +Parkin siRNA group increased significantly, and the secretion of IL-6 and TGF- beta in the hyperglycemic +Parkin overexpressed group decreased significantly (P0.05), indicating that Parkin could inhibit the high glucose induced renal tubule cell IL-6 and TGF- beta secretion. Three The expression of kin gradually decreases with the aggravation of DN renal interstitial damage, and is closely related to the structural and functional damage of DN renal tissue and the stress senescence of renal tubular cells. Parkin can inhibit the stress senescence of renal tubular epithelial cells induced by high glucose and the secretion of proinflammatory fibrotic factors.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

【参考文献】