RIP1和RIP3在糖尿病肾病大鼠肾组织中的表达及意义

发布时间：2018-05-04 18:04

本文选题：糖尿病肾病 + 坏死性凋亡　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:构建糖尿病肾病大鼠模型,探讨坏死性凋亡信号途径标志物RIP1和RIP3在该病大鼠模型肾组织中的表达及意义。方法:雄性成年SD大鼠,标准化喂养。实验分为糖尿病肾病模型组(DN)和正常对照组(NC),每组2、4、8、12、16周5个观察点,每个时点6只大鼠,DN组采用链脲菌素(STZ)腹腔注射造模,50只SD大鼠注射STZ,2周时成模40只,未成模7只,死亡3只。处死前采取血液、尿液标本,检测血糖、血清肌酐、血清尿素和24小时尿蛋白;肾脏石蜡切片作苏木素-伊红(Hematoxylin-eosin,HE)和过碘酸-雪夫(Periodic Acid-Schiff,PAS)染色普通病理;运用免疫组化、免疫荧光和蛋白印迹(Western Blot,WB)测定肾脏组织中受体交互作用蛋白1(Receptorinteraction protein1,RIP1)、受体交互作用蛋白3(Receptorinteraction protein 3,RIP3)及核转录因子Kappa B(Nuclearfactor-kappa B,NF-κB)的蛋白表达,实时荧光定量聚合酶链反应(Real-Time polymerase chain reaction,RT-PCR)方法测定肾组织的RIP1、RIP3及NF-κBm RNA相对表达量。分析各指标之间的相关性。结果:1.血、尿指标:1)血糖:(1)DN组不同时间点血糖比NC组明显增加(P0.01)。(2)DN组血糖随时间不断增加,差异有统计学意义(P0.05或P0.01)。2)24小时尿蛋白:(1)DN组不同时间点尿蛋白比NC组明显增加(P0.01)。(2)DN组24小时尿蛋白定量随时间不断增加,差异有统计学意义(P0.01)。3)血清肌酐及尿素:(1)DN组不同时间点血清肌酐及尿素比NC组明显增加(P0.01)。(2)DN组血清肌酐及尿素氮差异均无统计学意义(P0.05)。2.肾脏病理:(1)DN组大鼠肾脏发白、肿大,可见不同程度的肾小球肥大、细胞数增多、系膜基质增多、部分肾小管扩张及空泡样变性。(2)DN组不同时间点肾重指数比NC组明显增加(P0.01)。(3)DN组肾重指数随时间不断增加,差异有统计学意义(P0.01或P0.05)。3.免疫组化及荧光:(1)RIP1、RIP3及NF-κB主要表达于肾小球中;(2)DN组RIP1、RIP3及NF-κB不同时间点比NC组均明显增加(P0.01)。(3)DN组以上三个指标表达量不同时间点比较,均为2W与4W相比差异无统计学意义,8W、12W、16W显著高2W、4W,差异有统计学意义(P0.01),8W与12W相比差异无统计学意义,16W显著高于8W、12W,差异有统计学意义(P0.01或P0.05)。(4)DN组NF-κB的蛋白表达量与RIP1、RIP3呈正相关性;RIP1、RIP3与尿蛋白、肾重指数、血清肌酐及尿素均呈正相关。4.蛋白印迹:(1)DN组不同时间点RIP1、RIP3及NF-κB蛋白表达均高于NC组(P0.01)。(2)DN组以上三个指标表达量不同时间点比较,均为2W与4W相比差异无统计学意义,8W、12W、16W显著高于2W、4W,差异有统计学意义(P0.01),12W与8W相比差异无统计学意义,16W显著高于8W和12W,差异有统计学意义(P0.01)。(3)DN组NF-κB与RIP1、RIP3呈正相关性;RIP1、RIP3与尿蛋白、肾重指数、肌酐和尿素均呈正相关。5.Real-Time PCR:(1)DN组不同时间点RIP1、RIP3及NF-κB m RNA表达均高于NC组(P0.01),差异有统计学意义(P0.01)。(2)DN组以上三个指标表达量不同时间点比较,均为2W与4W差异无统计学意义,8W、12W、16W显著高于2W、4W,差异有统计学意义(P0.01),12W与8W相比差异无统计学意义,16W显著高于8W和12W,差异有统计学意义(P0.01)。(3)DN组NF-κB与RIP1、RIP3呈正相关性;RIP1、RIP3与尿蛋白、肾重指数均呈正相关,RIP3与肌酐和尿素呈正相关,RIP1与肌酐和尿素无明显相关性。结论:1.在糖尿病肾肾病大鼠肾组织中,坏死性途径凋亡可能被激活,可能与糖尿病肾病大鼠肾组织炎症反应和损伤有关。2.NF-κB和坏死性凋亡途径相互作用可能促进炎症反应和糖尿病肾病大鼠的肾组织损伤。
[Abstract]:Objective: to construct a diabetic nephropathy rat model and explore the expression and significance of RIP1 and RIP3 in the renal tissue of the rat model of necrotic apoptosis signal. Methods: male adult SD rats, standardized feeding. The experimental group was divided into diabetic nephropathy model group (DN) and normal group (NC), each group of 2,4,8,12,16 week 5 observation points, each time point. In 6 rats, group DN was intraperitoneally injected with streptozotocin (STZ), 50 SD rats were injected with STZ, 40 in 2 weeks, 7 without model, and 3 dead. Before death, blood, urine specimens, blood glucose, serum creatinine, serum urea and 24 hours proteinuria were detected, and renal paraffin section was made of Hematoxylin-eosin, HE and periodate - Schiff (P). Eriodic Acid-Schiff, PAS) staining common pathology, using immunohistochemistry, immunofluorescence and Western Blot (WB) for the determination of receptor interaction protein 1 (Receptorinteraction protein1, RIP1) in renal tissue, receptor interaction protein 3 (Receptorinteraction protein 3, RIP3) and nuclear transcription factors. The protein expression of - kappa B, the real-time fluorescent quantitative polymerase chain reaction (Real-Time polymerase chain reaction, RT-PCR) method for determining the relative expression of RIP1, RIP3 and NF- kappa Bm in renal tissue. Analysis of the correlation between the indexes. Results: 1. blood, urine index: 1) blood glucose: (1) blood glucose at different time points in the DN group was significantly higher than that of the group. (2) blood group blood The sugar was increased with time, the difference was statistically significant (P0.05 or P0.01).2) 24 hour urine protein: (1) the urine protein in the DN group was significantly increased at different time points (P0.01). (2) the 24 hour urine protein in the DN group increased with time, the difference was statistically significant (P0.01).3) serum creatinine and urea: (1) the serum creatinine and urea at different time points in the DN group Compared with group NC (P0.01), (2) there was no significant difference in serum creatinine and urea nitrogen in group DN (P0.05).2. renal pathology: (1) kidney white and swelling in group DN, the number of glomerular cells increased, the number of cells increased, the mesangial matrix increased, partial renal tubule dilation and vacuolating degeneration. (2) the renal weight index of group DN at different time points was compared with that of the NC group (3) (3) the renal weight index of group DN increased with time, the difference was statistically significant (P0.01 or P0.05).3. immunization and fluorescence: (1) RIP1, RIP3 and NF- kappa B were mainly expressed in the glomeruli; (2) DN RIP1, RIP3 and different time points were significantly increased. (3) the expression of three indicators at different time points above The difference between 2W and 4W was not statistically significant, 8W, 12W, 16W were significantly higher 2W, 4W, and the difference was statistically significant (P0.01). The difference between 8W and 12W was not statistically significant. (4) the difference was statistically significant. The index, serum creatinine and urea were positively correlated with.4. blot: (1) the expression of RIP1, RIP3 and NF- kappa B protein at different time points in group DN were higher than that of NC group (P0.01). (2) the differences between 2W and 4W were not statistically significant compared with those of 2W and 4W. There was no statistical significance compared with 8W, 16W was significantly higher than 8W and 12W (P0.01). (3) there was a positive correlation between NF- kappa B and RIP1, RIP3 in DN group; RIP1, RIP3 and urine protein, renal weight index, creatinine and urea were positively correlated (1) There were statistical significance (P0.01). (2) the difference between 2W and 4W was not statistically significant, 8W, 12W, 16W were significantly higher than 2W, 4W, and the difference was statistically significant (P0.01). There was no statistical significance in 12W and 8W. (3) the difference was statistically significant. IP3 has positive correlation; RIP1, RIP3 and urine protein, renal weight index are positively correlated, RIP3 is positively correlated with creatinine and urea, RIP1 has no significant correlation with creatinine and urea. Conclusion: 1. in diabetic renal nephrotic rats, the apoptosis of necrotic pathway may be activated, which may be related to the inflammatory reaction and injury of renal tissue in diabetic nephropathy rats, which may be related to.2 The interaction between.NF- B and necrotic apoptotic pathway may promote inflammatory reaction and renal tissue damage in diabetic nephropathy rats.

【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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