發(fā)布時(shí)間：2018-04-30 15:12

  本文選題：糖尿病腎病 + 應(yīng)激性衰老�。� 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：糖尿病腎病(Diabetic Nephropathy,DN)是糖尿病的主要并發(fā)癥之一,是目前世界范圍內(nèi)導(dǎo)致終末期腎臟病及透析的首要病因。DN發(fā)病機(jī)制復(fù)雜,其中腎小管上皮細(xì)胞應(yīng)激性衰老是導(dǎo)致DN發(fā)生發(fā)展的重要機(jī)制。研究證實(shí),腎小管細(xì)胞衰老是DN病理?yè)p傷標(biāo)志之一。衰老腎小管細(xì)胞能夠產(chǎn)生大量促炎因子和促纖維化因子等衰老相關(guān)分泌表型,引起組織局部炎癥反應(yīng)和纖維化。同時(shí)抑制腎小管的加速衰老可延緩DN進(jìn)展為終末期腎臟疾病。E3泛素連接酶Parkin在腦、心、腎等組織器官中均可廣泛表達(dá),其基因突變與老年性癡呆、帕金森病的發(fā)病密切相關(guān)。過(guò)表達(dá)Parkin能夠延長(zhǎng)果蠅壽限,提示Parkin可能是一個(gè)潛在的抗衰老因子,但目前Parkin與DN腎臟衰老關(guān)系尚不清楚。本研究旨在探討Parkin在DN誘導(dǎo)的腎小管細(xì)胞應(yīng)激性衰老中的作用,為DN的防治尋找新的有效靶點(diǎn)和理論依據(jù)。一、實(shí)驗(yàn)方法1.臨床病例研究對(duì)象為2015年1月至2016年6月期間于我科就診的2型糖尿病患者。行腎臟病理確診為DN共40例。對(duì)照組來(lái)源于腎臟原發(fā)性腫瘤患者行切除術(shù)后遠(yuǎn)端正常腎組織,并行腎組織病理檢查,共10例。收集兩組基線臨床數(shù)據(jù),包括年齡、性別、身高、體重、體質(zhì)指數(shù)、收縮壓、舒張壓。于腎活檢或手術(shù)1天前收集患者血尿標(biāo)本,-80℃超低溫保存,測(cè)定其生化指標(biāo)包括:血肌酐、血清尿素氮、血糖、糖化血紅蛋白、血清胱抑C、血清白蛋白、血尿酸、血清總膽固醇、甘油三酯、高密度脂蛋白、低密度脂蛋白、C反應(yīng)蛋白、尿蛋白定量、尿N-乙酰-β-氨基葡萄糖苷酶和估算腎小球?yàn)V過(guò)率。免疫組化檢測(cè)糖尿病腎病患者腎組織Parkin的表達(dá),顯微鏡下觀察并計(jì)算Parkin陽(yáng)性率,并與臨床資料和病理?yè)p傷評(píng)分進(jìn)行相關(guān)性分析。免疫熒光檢測(cè)糖尿病腎病患者腎組織Parkin分別與衰老標(biāo)志物p16、衰老相關(guān)分泌表型IL-6和TGF-β的關(guān)系。2.體外實(shí)驗(yàn)剪取3-4周齡的C57雄性鼠雙腎皮質(zhì),經(jīng)膠原酶孵育及雙重篩網(wǎng)濾過(guò)后獲得腎小管細(xì)胞。采用原代腎小管細(xì)胞專用培養(yǎng)基,常規(guī)培養(yǎng)兩代后用于后續(xù)實(shí)驗(yàn)。通過(guò)30mmol/l高糖刺激腎小管細(xì)胞72h,并通過(guò)免疫熒光檢測(cè)腎小管細(xì)胞p16、γ-H2AX的陽(yáng)性率,WB檢測(cè)p16和Parkin蛋白水平的變化,PCR檢測(cè)Parkin mRNA水平的變化,ELISA檢測(cè)細(xì)胞培養(yǎng)基上清中IL-6和TGF-β的分泌。進(jìn)一步采用Parkin過(guò)表達(dá)腺病毒或Parkin siRNA轉(zhuǎn)染腎小管細(xì)胞,檢測(cè)Parkin上調(diào)或下調(diào)后高糖刺激的腎小管細(xì)胞p16、γ-H2AX的表達(dá)變化,及IL-6和TGF-β的分泌。二、結(jié)果1.糖尿病腎病腎組織Parkin的表達(dá)變化及與腎小管細(xì)胞應(yīng)激性衰老、腎間質(zhì)損傷的關(guān)系免疫組化顯示,Parkin主要表達(dá)于腎小管上皮細(xì)胞。對(duì)照組腎小管細(xì)胞Parkin陽(yáng)性率為43.5±6.0%,IFTA 0分組腎小管細(xì)胞Parkin陽(yáng)性率與對(duì)照組相近,為40.8±4.3%,IFTA 1分組為32.8±4.8%,IFTA 2分組為28.5±4.2%,IFTA 3分組為19.0±4.9%,提示腎小管細(xì)胞Parkin的陽(yáng)性率隨著DN的進(jìn)展逐漸降低。相關(guān)性分析顯示Parkin的陽(yáng)性率與腎小球硬化、腎小管萎縮與腎間質(zhì)纖維化、腎間質(zhì)炎癥均呈顯著負(fù)相關(guān)(P0.05),與腎臟功能損傷指標(biāo),包括尿蛋白定量、尿NAG、尿素氮、血清胱抑素C、血肌酐和收縮壓均呈負(fù)相關(guān),與e GFR呈正相關(guān)(P0.05)。表明Parkin表達(dá)降低與DN腎結(jié)構(gòu)和功能損傷水平均密切相關(guān)。進(jìn)一步通過(guò)免疫熒光檢測(cè)糖尿病腎病患者腎組織腎小管細(xì)胞Parkin分別與p16、IL-6及TGF-β的關(guān)系發(fā)現(xiàn),與對(duì)照組相比,DN患者腎小管細(xì)胞衰老標(biāo)志物p16、促炎性因子IL-6、促纖維化因子TGF-β的表達(dá)增多,與Parkin的表達(dá)趨勢(shì)相反。2.Parkin與DN腎小管上皮細(xì)胞應(yīng)激性衰老的體外研究2.1高糖誘導(dǎo)腎小管細(xì)胞應(yīng)激性衰老體外研究發(fā)現(xiàn),與正常對(duì)照組相比,高糖組腎小管細(xì)胞p16和γ-H2AX的陽(yáng)性率顯著增大(P0.05);WB結(jié)果表明高糖組p16蛋白表達(dá)水平為對(duì)照組的2.13倍(P0.05);ELISA表明腎小管細(xì)胞IL-6、TGF-β的分泌顯著增多(P0.05),提示高糖環(huán)境可誘導(dǎo)腎小管細(xì)胞應(yīng)激性衰老及IL-6、TGF-β的分泌。2.2高糖刺激下腎小管細(xì)胞Parkin的表達(dá)變化與對(duì)照組相比,高糖刺激后腎小管細(xì)胞Parkin的mRNA水平下降了60.8%,蛋白水平則下降了53.1%(P0.05),與DN患者中Parkin表達(dá)逐漸減少的趨勢(shì)相同,提示高糖可抑制腎小管細(xì)胞Parkin的表達(dá)。2.3.Parkin對(duì)高糖誘導(dǎo)的腎小管細(xì)胞應(yīng)激性衰老的作用Parkin過(guò)表達(dá)腺病毒轉(zhuǎn)染上調(diào)Parkin的表達(dá)后發(fā)現(xiàn),與高糖組比較,Parkin過(guò)表達(dá)后腎小管細(xì)胞p16和γ-H2AX的陽(yáng)性率分別減少了32.1%和37.8%(P0.05),WB提示該組p16蛋白水平減少了18.4%(P0.05,vs高糖組);Parkin siRNA沉默Parkin發(fā)現(xiàn),高糖刺激后,Parkin干擾組p16和γ-H2AX的陽(yáng)性腎小管細(xì)胞比例較單純高糖刺激組升高了24.5%和17.6%(P0.05);WB提示該組p16蛋白水平升高了21.0%(P0.05,vs高糖組),表明Parkin可抑制高糖誘導(dǎo)的腎小管細(xì)胞應(yīng)激性衰老。進(jìn)一步檢測(cè)細(xì)胞培養(yǎng)基上清IL-6和TGF-β的分泌,發(fā)現(xiàn)高糖+Parkin siRNA組腎小管細(xì)胞IL-6和TGF-β的分泌較高糖組均顯著增強(qiáng),同時(shí)高糖+Parkin過(guò)表達(dá)組IL-6和TGF-β的分泌較高糖組顯著減少(P0.05),表明Parkin可抑制高糖誘導(dǎo)的腎小管細(xì)胞IL-6、TGF-β的分泌。三、結(jié)論E3泛素連接酶Parkin的表達(dá)隨著DN腎間質(zhì)損傷的加重而逐漸減少,并與DN腎組織結(jié)構(gòu)與功能損傷、腎小管細(xì)胞應(yīng)激性衰老密切相關(guān);Parkin可抑制高糖誘導(dǎo)的腎小管上皮細(xì)胞應(yīng)激性衰老和促炎促纖維化因子的分泌。
[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.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R587.2;R692.9

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