本文選題：糖尿病腎病 + 腎小管上皮細胞　； 參考：《第三軍醫(yī)大學》2015年碩士論文

【摘要】：研究背景與目的糖尿病腎病(Diabetic nephropathy,DN)是導致終末期腎病(enD stage renal Disease,ESRD)最主要的原因之一。關于DN進展機制尚未完全闡明,近年研究認為腎小管間質(zhì)損傷發(fā)揮重要作用,與DN預后直接相關。而腎小管上皮細胞衰老作為腎小管間質(zhì)損傷重要的細胞生物學事件,在DN進展中的作用受到廣泛關注和重視。DN腎小管上皮細胞衰老是不依賴于增齡的衰老過程,它是在糖尿病所致的糖脂代謝紊亂、持續(xù)性氧應激以及慢性炎癥等病理性微環(huán)境下發(fā)生的應激性早衰(stress inDuceD premature senescence,SIPS)。SIPS是不依賴增齡的細胞加速衰老,表現(xiàn)為細胞周期不可逆停滯和增殖能力永久性喪失,導致組織細胞再生和修復能力減退,器官功能障礙。衰老細胞除了SA-β-gal、p16、p21等特異性分子標志外,還可釋放促炎因子、炎癥因子等衰老相關分泌表型(senescence associateD secretory phenotype,SASP)損害周邊正常細胞,導致局部組織細胞炎癥與纖維化。此外,衰老細胞還具有凋亡抵抗特性可逃逸免疫細胞清除,導致衰老細胞堆積滯留,損害效應遷延放大,進而加速疾病進展。研究表明衰老細胞凋亡抵抗的原因在于誘騙受體(Decoy receptor2,DcR2)高表達。衰老成纖維細胞因高表達DcR2而逃逸細胞凋亡,可導致肝臟組織細胞纖維化與肝功能受損;前列腺癌等腫瘤細胞因DcR2表達水平上調(diào)而呈現(xiàn)凋亡抵抗,導致鄰近正常細胞惡變和腫瘤細胞轉移。此外,在DN大鼠模型中發(fā)現(xiàn)DcR2高表達可抑制腎小管上皮細胞凋亡。而關于DN進展中DcR2表達變化是否與腎組織損傷及腎功能之間有關,以及DcR2表達變化與衰老腎小管上皮細胞之間的關系目前尚不清楚。本研究首先分析DN腎小管上皮細胞DcR2、p16表達變化與腎組織損傷程度及腎功能之間的關系,然后研究DcR2表達與衰老腎小管上皮細胞之間的關系,并明確衰老腎小管細胞是否呈現(xiàn)凋亡抵抗表型。通過上述研究,以期明確DcR2在DN進展中具有重要作用,并與衰老腎小管上皮細胞凋亡抵抗密切相關。以為DN早期干預衰老腎小管細胞尋找關鍵靶分子,并從靶向清除衰老細胞角度提高DN救治水平和改善腎臟預后奠定理論基礎。研究方法1.臨床資料:選取2010-2013年在第三軍醫(yī)大學大坪醫(yī)院腎內(nèi)科住院的2型糖尿病患者36例,并經(jīng)腎活檢病理檢查診斷為Dn。根據(jù)腎小管間質(zhì)損傷程度分為早期Dn和進展期Dn,各18例。對照組腎組織標本為在我院住院的腎錯構瘤或腎臟腫瘤切除術病灶旁組織經(jīng)病理檢查為正常的組織。采集各組患者的臨床資料及相關實驗室檢查結果,根據(jù)ckD-epi公式計算腎小球濾過率(estimateDglomerularfiltration,egfr)。2.腎組織損傷程度評分與分級標準:根據(jù)2010年《美國腎臟病雜志》發(fā)表的新型病理分級系統(tǒng)診斷標準進行評分。按照腎小管萎縮和代償性擴張、腎間質(zhì)纖維化、間質(zhì)炎癥程度將Dn分為早期Dn(earlyDn)和進展期Dn(progressiveDn)。3.檢測指標與方法3.1免疫組化檢測Dcr2、p16表達;3.2免疫熒光檢測Dcr2、p16、flip、caspase-3表達;3.3利用激光共聚焦觀察Dcr2與p16、flip、caspase-3之間共表達情況。4.結果判讀方法結果的判讀采用雙盲法,由兩名與本研究無關人員判讀。每張載玻片連續(xù)計數(shù)10個高倍視野下相關指標陽性表達的腎小管上皮細胞(胞核表達)或是腎小管(胞漿表達),并計算陽性表達的腎小管上皮細胞或腎小管占腎小管細胞總數(shù)的百分比。共聚焦顯微鏡下計數(shù)Dcr2與p16、Dcr2與flip、Dcr2與caspase-3雙陽性腎小管數(shù)及各指標單陽性腎小管數(shù)占腎小管總數(shù)百分比。5.統(tǒng)計學分析采用spss18.0統(tǒng)計軟件處理,計量資料以sx±表示。Dn患者的臨床資料及p16、Dcr2陽性表達百分率與對照組比較,正態(tài)分布資料采用t檢驗,非正態(tài)分布資料比較采用秩和檢驗。腎組織p16、Dcr2陽性表達百分率與臨床資料采用pearson相關分析,與腎組織病理損傷程度評分采用spearman相關分析。p0.05表示有統(tǒng)計學意義。結果1.36例Dn患者臨床資料特征36例Dn患者中男性17例,女性19例。與對照組相比,Dn組患者收縮壓、尿蛋白定量、尿nag、尿蛋白/肌酐、糖化血紅蛋白、血肌酐、光抑素c、尿素氮、甘油三酯顯著增高,egfr顯著降低(p0.05)。Dn組同對照組年齡、性別、bmi、舒張壓、尿酸、c反應蛋白、總膽固醇、高密度脂蛋白、低密度脂蛋白相比差異無統(tǒng)計學意義(p0.05)。2.Dn腎組織Dcr2、p16表達與臨床資料及腎組織損傷評分的相關性Dn中Dcr2只表達于腎小管上皮細胞,p16可表達于腎小球細胞、腎小管上皮細胞與腎間質(zhì)細胞。正常對照組腎組織p16、Dcr2表達水平低下。腎小管上皮細胞Dcr2、p16陽性表達百分率同對照組相比顯著增高,且進展期Dn腎小管細胞上皮Dcr2、p16表達水平較早期Dn明顯增加。Dn患者腎組織腎小管上皮細胞Dcr2、p16表達量與收縮壓、尿蛋白定量、尿nag、尿蛋白/肌酐、甘油三脂、尿素氮、血肌酐、光抑素c呈正相關,與egfr呈負相關,而與年齡、舒張壓、bmi、白蛋白、糖化血紅蛋白、膽固醇、尿酸、低密度脂蛋白、高密度脂蛋白、c反應蛋白無相關性。Dn患者腎組織腎小管上皮細胞Dcr2、p16表達量與腎組織系膜增生、腎小球硬化、腎間質(zhì)炎癥、腎小管萎縮與間質(zhì)纖維化病變程度顯著相關。3.Dn腎組織Dcr2與p16的共表達情況激光共聚焦示Dn腎組織中Dcr2與p16共表達于同一腎小管細胞,共表達率隨著腎小管間質(zhì)損傷的評分升高而增加,且Dcr2表達量較p16少�？梢娦〔糠謕16與Dcr2單陽性腎小管細胞,且p16單陽性腎小管細胞百分率較Dcr2單陽性腎小管細胞百分率高,Dn各組p16單陽性腎小管細胞百分率較對照明顯增高,Dcr2單陽性腎小管細胞在各個組間比較無顯著差異。4.Dn腎組織Dcr2與凋亡相關標志的共表達情況激光共聚焦示Dcr2與抗凋亡分子flip共表達于同一腎小管細胞,共表達率隨著腎小管間質(zhì)損傷的評分升高而增加�？梢娦〔糠謋lip單陽性腎小管細胞,其百分率較Dcr2單陽性腎小管百分率高,flip單陽性腎小管細胞百分率在各組間比較無顯著差異。免疫共染示Dcr2陽性的腎小管細胞不表達或低表達caspase-3。早期Dn中caspase-3單陽性腎小管細胞百分率較Dcr2單陽性腎小管細胞百分率低,其與對照相比無顯著差異。進展期Dn中caspase-3單陽性腎小管細胞百分率較對照組及早期Dn明顯增高。結論Dcr2與Dn腎小管上皮細胞衰老及其凋亡抵抗特性密切相關,在Dn腎組織損傷中發(fā)揮重要作用。
[Abstract]:Background and objective diabetic nephropathy (Diabetic nephropathy, DN) is one of the most important causes of enD stage renal Disease (ESRD). The mechanism of DN progression is not fully elucidated. In recent years, renal tubulointerstitial damage plays an important role, which is directly related to the prognosis of DN. The important cell biological events of renal tubulointerstitial injury, the role in the progress of DN is widely concerned and attached to the aging process of.DN renal tubular epithelial cells, which is not dependent on aging process. It is the stress induced premature failure in the pathological microenvironment, such as diabetes, glucose and lipid metabolism, persistent oxygen stress and chronic inflammation. (stress inDuceD premature senescence, SIPS).SIPS is not dependent on aging cells to accelerate senescence, manifested as irreversible stagnation of cell cycle and permanent loss of proliferative capacity, resulting in dysfunctional tissue regeneration and repair, and organ dysfunction. Senescent cells can also be released in addition to specific molecular markers such as SA- beta -gal, p16, p21, etc. The aging related secretory phenotypes such as inflammatory factors and inflammatory factors (senescence associateD secretory phenotype, SASP) damage peripheral normal cells and cause inflammation and fibrosis in local tissue cells. In addition, aging cells also have apoptosis resistance characteristics that can escape immune cell clearance, cause accumulation and retention of senescent cells, damage effect enlargement and enlargement. The study shows that the apoptosis resistance of senescent cells is due to the high expression of Decoy receptor2 (DcR2). Aging fibroblasts escape apoptosis due to high expression of DcR2, which can lead to liver tissue fibrosis and liver function damage, and prostate cancer cells are apoptotic due to the up regulation of DcR2 expression level. Resistance, leading to adjacent normal cell malignancy and tumor cell metastasis. In addition, the high expression of DcR2 can inhibit the apoptosis of renal tubular epithelial cells in the DN rat model. The relationship between the changes of DcR2 expression in the progression of DN and the renal tissue injury and renal function, and the relationship between the changes of DcR2 expression and the epithelial cells of senile renal tubule This study first analyzed the relationship between the changes of DcR2, p16 expression, the degree of renal tissue injury and renal function in DN renal tubular epithelial cells, and then studied the relationship between the expression of DcR2 and the tubular epithelial cells of the aged renal tubules, and the expression of apoptosis resistance phenotype in the aging renal tubule cells. DN has an important role in progresses and is closely related to the apoptosis resistance of tubular epithelial cells in aging renal tubules. It is thought that early intervention of DN in aging renal tubule cells to find the key target molecules, and to improve the level of DN treatment and improve the prognosis of kidney from the angle of targeting senescence cells to improve the prognosis of kidney. Research method 1. clinical data: select 2010-2013 years in the first place. 36 cases of type 2 diabetes hospitalized in the nephrology department of Daping Hospital of the Third Army Medical University were diagnosed as Dn. based on renal tubulointerstitial damage and divided into early Dn and progressive Dn, each 18 cases. The clinical data of the patients in each group and the results of the related laboratory examination were collected and the glomerular filtration rate (estimateDglomerularfiltration, EGFR).2. renal tissue injury degree score and grading standard were calculated according to the ckD-epi formula. According to the new type of pathological grading system published in the American Journal of kidney disease in 2010, the score was scored. Renal tubule atrophy and compensatory dilation, renal interstitial fibrosis and interstitial inflammation, Dn was divided into early Dn (earlyDn) and progressing Dn (progressiveDn).3. detection index and method 3.1 immunohistochemical detection Dcr2, p16 expression, and 3.2 immunofluorescence detection Dcr2, p16, flip, caspase-3 expression; 3.3 A double blind method was used for the interpretation of the results of the.4. result interpretation. Two consecutive slides were used to count the renal tubular epithelial cells (nucleus expression) or renal tubules (cytoplasm of the renal tubules) that were positive in 10 high times of high field of vision, and the positive expression of renal tubular epithelial cells was calculated. The percentage of the total number of renal tubules in the renal tubules. The number of Dcr2 and p16, Dcr2 and flip, the number of Dcr2 and caspase-3 double positive renal tubules and the number of single positive renal tubules in the percentage of the total renal tubules were analyzed by the confocal microscopy, and the statistical analysis of the total number of renal tubules was analyzed by SPSS18.0 statistical software, and the measurement data were expressed as SX + in the clinical data of the.Dn patients. The percentage of positive expression of p16 and Dcr2 was compared with that of the control group. The normal distribution data were tested by t test. The non normal distribution data were compared with the rank sum test. The renal tissue p16, the percentage of Dcr2 positive expression and the clinical data were correlated with the Pearson analysis, and the Spearman correlation analysis.P0.05 was statistically significant for the degree score of renal pathological injury. Results the clinical data of 1.36 patients with Dn were characterized by 17 men and 19 women in 36 cases. Compared with the control group, the systolic pressure, urine protein, urine NAG, urine protein / creatinine, glycosylated hemoglobin, creatinine, serum creatinine C, urea nitrogen, triglycerides were significantly increased in the group Dn, and EGFR significantly decreased (P0.05).Dn group with the control age, sex, BMI, diastolic. Pressure, uric acid, C reactive protein, total cholesterol, high density lipoprotein and low density lipoprotein, there is no significant difference (P0.05).2.Dn renal tissue Dcr2, p16 expression and clinical data and renal tissue damage score in Dn Dcr2 only expressed in renal tubular epithelial cells, p16 can be expressed in glomerular cells, renal tubular epithelial cells and renal interstitium In normal control group, the expression of p16 and Dcr2 in renal tissue was low. The positive expression of Dcr2, p16 in renal tubular epithelial cells was significantly higher than that in the control group, and the Dcr2 in the epithelial cells of Dn renal tubule cells in the progressing stage, the expression level of p16 in the renal tubule was significantly increased in.Dn patients' renal tubular upper cutaneous cell Dcr2, p16 expression and systolic pressure, urine protein determination. Quantity, urine NAG, urine protein / creatinine, glycerol three fat, urea nitrogen, serum creatinine, and C were positively correlated with EGFR, but with age, diastolic pressure, BMI, albumin, glycated hemoglobin, cholesterol, uric acid, LDL, HDL, C anti protein,.Dn patients with no correlation of Dcr2, p16 expression and p16 expression Renal tissue mesangial hyperplasia, glomerulosclerosis, renal interstitial inflammation, renal tubule atrophy and interstitial fibrosis, the co expression of Dcr2 and p16 in.3.Dn renal tissue, the co expression of Dcr2 and p16 in the same renal tubule cells in Dn renal tissue, the co expression rate increases with the score of renal tubulointerstitial injury, and Dcr The expression of 2 was less than that of p16. A small portion of p16 and Dcr2 single positive renal tubule cells were found, and the percentage of p16 single positive renal tubule cells was higher than that of Dcr2 positive renal tubule cells. The percentage of p16 single positive renal tubular cells in each group of Dn was significantly higher than that of the control. There was no significant difference in.4.Dn renal tissue Dc between the Dcr2 single positive renal tubule cells in each group. Co expression of R2 and apoptosis related markers was co expressed by laser confocal Dcr2 and anti apoptotic molecule flip in the same renal tubule cells. The co expression rate increased with the increase of renal tubulointerstitial damage score. The percentage of small part of flip single positive renal tubule cells was higher than that of Dcr2 positive renal tubules and flip single positive renal tubules. There was no significant difference between the percentages of cells in each group. The percentage of Dcr2 positive renal tubule cells that did not express or low expression of caspase-3. in the early Dn was lower than the percentage of Dcr2 positive renal tubule cells in the early stage of caspase-3., and there was no significant difference between the percentage of single positive renal tubular cells in the Dcr2 positive renal tubule cells. The percentage of cell cell was significantly higher than that of the control group and the early Dn. Conclusion Dcr2 and Dn renal tubular epithelial cell aging and apoptosis resistance characteristics are closely related, and play an important role in the injury of Dn renal tissue.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R587.2

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6 楊曉;張春;鄧安國;朱忠華;;結締組織生長因子在腎小管上皮細胞細胞外基質(zhì)積聚中的作用[A];“中華醫(yī)學會腎臟病學分會2004年年會”暨“第二屆全國中青年腎臟病學術會議”論文匯編[C];2004年

7 張瑜;周建華;;肝素對腎小管上皮細胞膜結合補體調(diào)節(jié)蛋白表達的影響及意義[A];中華醫(yī)學會第十四次全國兒科學術會議論文匯編[C];2006年

8 楊雅麗;程曉霞;;腎小管上皮細胞凋亡在腎間質(zhì)纖維化中的作用及其防治的研究進展[A];2009全國時間生物醫(yī)學學術會議論文集[C];2009年

9 譚玉莉;黃妍;楊亦彬;;魚膽汁毒素致腎小管上皮細胞死亡方式及實施干預的試驗研究[A];貴州省醫(yī)學會腎臟病學分會2008年學術年會論文匯編[C];2008年

10 喬f^;陳香美;吳鏑;丁瑞;師鎖柱;謝院生;洪權;呂楊;王兆霞;尹忠;;衰老大鼠腎臟缺血/再灌注損傷中腎小管上皮細胞不同凋亡途徑的研究[A];“中華醫(yī)學會腎臟病學分會2004年年會”暨“第二屆全國中青年腎臟病學術會議”論文匯編[C];2004年

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