DcR2在糖尿病肾病肾小管上皮细胞的表达与意义

发布时间：2018-06-16 00:45

本文选题：糖尿病肾病 + 肾小管上皮细胞　；参考：《第三军医大学》2015年硕士论文

【摘要】：研究背景与目的糖尿病肾病(Diabetic nephropathy,DN)是导致终末期肾病(enD stage renal Disease,ESRD)最主要的原因之一。关于DN进展机制尚未完全阐明,近年研究认为肾小管间质损伤发挥重要作用,与DN预后直接相关。而肾小管上皮细胞衰老作为肾小管间质损伤重要的细胞生物学事件,在DN进展中的作用受到广泛关注和重视。DN肾小管上皮细胞衰老是不依赖于增龄的衰老过程,它是在糖尿病所致的糖脂代谢紊乱、持续性氧应激以及慢性炎症等病理性微环境下发生的应激性早衰(stress inDuceD premature senescence,SIPS)。SIPS是不依赖增龄的细胞加速衰老,表现为细胞周期不可逆停滞和增殖能力永久性丧失,导致组织细胞再生和修复能力减退,器官功能障碍。衰老细胞除了SA-β-gal、p16、p21等特异性分子标志外,还可释放促炎因子、炎症因子等衰老相关分泌表型(senescence associateD secretory phenotype,SASP)损害周边正常细胞,导致局部组织细胞炎症与纤维化。此外,衰老细胞还具有凋亡抵抗特性可逃逸免疫细胞清除,导致衰老细胞堆积滞留,损害效应迁延放大,进而加速疾病进展。研究表明衰老细胞凋亡抵抗的原因在于诱骗受体(Decoy receptor2,DcR2)高表达。衰老成纤维细胞因高表达DcR2而逃逸细胞凋亡,可导致肝脏组织细胞纤维化与肝功能受损;前列腺癌等肿瘤细胞因DcR2表达水平上调而呈现凋亡抵抗,导致邻近正常细胞恶变和肿瘤细胞转移。此外,在DN大鼠模型中发现DcR2高表达可抑制肾小管上皮细胞凋亡。而关于DN进展中DcR2表达变化是否与肾组织损伤及肾功能之间有关,以及DcR2表达变化与衰老肾小管上皮细胞之间的关系目前尚不清楚。本研究首先分析DN肾小管上皮细胞DcR2、p16表达变化与肾组织损伤程度及肾功能之间的关系,然后研究DcR2表达与衰老肾小管上皮细胞之间的关系,并明确衰老肾小管细胞是否呈现凋亡抵抗表型。通过上述研究,以期明确DcR2在DN进展中具有重要作用,并与衰老肾小管上皮细胞凋亡抵抗密切相关。以为DN早期干预衰老肾小管细胞寻找关键靶分子,并从靶向清除衰老细胞角度提高DN救治水平和改善肾脏预后奠定理论基础。研究方法1.临床资料:选取2010-2013年在第三军医大学大坪医院肾内科住院的2型糖尿病患者36例,并经肾活检病理检查诊断为Dn。根据肾小管间质损伤程度分为早期Dn和进展期Dn,各18例。对照组肾组织标本为在我院住院的肾错构瘤或肾脏肿瘤切除术病灶旁组织经病理检查为正常的组织。采集各组患者的临床资料及相关实验室检查结果,根据ckD-epi公式计算肾小球滤过率(estimateDglomerularfiltration,egfr)。2.肾组织损伤程度评分与分级标准:根据2010年《美国肾脏病杂志》发表的新型病理分级系统诊断标准进行评分。按照肾小管萎缩和代偿性扩张、肾间质纤维化、间质炎症程度将Dn分为早期Dn(earlyDn)和进展期Dn(progressiveDn)。3.检测指标与方法3.1免疫组化检测Dcr2、p16表达;3.2免疫荧光检测Dcr2、p16、flip、caspase-3表达;3.3利用激光共聚焦观察Dcr2与p16、flip、caspase-3之间共表达情况。4.结果判读方法结果的判读采用双盲法,由两名与本研究无关人员判读。每张载玻片连续计数10个高倍视野下相关指标阳性表达的肾小管上皮细胞(胞核表达)或是肾小管(胞浆表达),并计算阳性表达的肾小管上皮细胞或肾小管占肾小管细胞总数的百分比。共聚焦显微镜下计数Dcr2与p16、Dcr2与flip、Dcr2与caspase-3双阳性肾小管数及各指标单阳性肾小管数占肾小管总数百分比。5.统计学分析采用spss18.0统计软件处理,计量资料以sx±表示。Dn患者的临床资料及p16、Dcr2阳性表达百分率与对照组比较,正态分布资料采用t检验,非正态分布资料比较采用秩和检验。肾组织p16、Dcr2阳性表达百分率与临床资料采用pearson相关分析,与肾组织病理损伤程度评分采用spearman相关分析。p0.05表示有统计学意义。结果1.36例Dn患者临床资料特征36例Dn患者中男性17例,女性19例。与对照组相比,Dn组患者收缩压、尿蛋白定量、尿nag、尿蛋白/肌酐、糖化血红蛋白、血肌酐、光抑素c、尿素氮、甘油三酯显著增高,egfr显著降低(p0.05)。Dn组同对照组年龄、性别、bmi、舒张压、尿酸、c反应蛋白、总胆固醇、高密度脂蛋白、低密度脂蛋白相比差异无统计学意义(p0.05)。2.Dn肾组织Dcr2、p16表达与临床资料及肾组织损伤评分的相关性Dn中Dcr2只表达于肾小管上皮细胞,p16可表达于肾小球细胞、肾小管上皮细胞与肾间质细胞。正常对照组肾组织p16、Dcr2表达水平低下。肾小管上皮细胞Dcr2、p16阳性表达百分率同对照组相比显著增高,且进展期Dn肾小管细胞上皮Dcr2、p16表达水平较早期Dn明显增加。Dn患者肾组织肾小管上皮细胞Dcr2、p16表达量与收缩压、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呈正相关,与egfr呈负相关,而与年龄、舒张压、bmi、白蛋白、糖化血红蛋白、胆固醇、尿酸、低密度脂蛋白、高密度脂蛋白、c反应蛋白无相关性。Dn患者肾组织肾小管上皮细胞Dcr2、p16表达量与肾组织系膜增生、肾小球硬化、肾间质炎症、肾小管萎缩与间质纤维化病变程度显著相关。3.Dn肾组织Dcr2与p16的共表达情况激光共聚焦示Dn肾组织中Dcr2与p16共表达于同一肾小管细胞,共表达率随着肾小管间质损伤的评分升高而增加,且Dcr2表达量较p16少。可见小部分p16与Dcr2单阳性肾小管细胞,且p16单阳性肾小管细胞百分率较Dcr2单阳性肾小管细胞百分率高,Dn各组p16单阳性肾小管细胞百分率较对照明显增高,Dcr2单阳性肾小管细胞在各个组间比较无显著差异。4.Dn肾组织Dcr2与凋亡相关标志的共表达情况激光共聚焦示Dcr2与抗凋亡分子flip共表达于同一肾小管细胞,共表达率随着肾小管间质损伤的评分升高而增加。可见小部分flip单阳性肾小管细胞,其百分率较Dcr2单阳性肾小管百分率高,flip单阳性肾小管细胞百分率在各组间比较无显著差异。免疫共染示Dcr2阳性的肾小管细胞不表达或低表达caspase-3。早期Dn中caspase-3单阳性肾小管细胞百分率较Dcr2单阳性肾小管细胞百分率低,其与对照相比无显著差异。进展期Dn中caspase-3单阳性肾小管细胞百分率较对照组及早期Dn明显增高。结论Dcr2与Dn肾小管上皮细胞衰老及其凋亡抵抗特性密切相关,在Dn肾组织损伤中发挥重要作用。
[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.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

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