c-FLIP在糖尿病肾病大鼠肾组织中的表达及意义

发布时间：2018-05-24 22:17

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:使用链脲佐菌素(STZ)构建糖尿病大鼠模型,探讨细胞型Fas相关死亡域蛋白样白介素-1β转换酶抑制蛋白(c-FLIP)在糖尿病肾病大鼠肾组织中的表达以及c-FLIP介导坏死性凋亡通路在糖尿病肾病发病机制中的作用。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),DN组采用链脲菌素(STZ)55mg/Kg腹腔注射造模,每组2、4、8、12、16周5个观察点,每个时点6只大鼠。处死前采取血液、尿液标本,检测血糖、血清肌酐、血清尿素氮和24小时尿蛋白定量;肾脏石蜡切片作普通病理;免疫组化、免疫印迹(WB)测定肾组织中c-FLIP、受体交互作用蛋白1(RIP1)和受体交互作用蛋白3(RIP3)表达,RT-PCR测定肾组织的c-FLIP、RIP1及RIP3 mRNA相对表达量。并分析各指标之间的相关性。结果:1.血、尿指标:1)血糖:DN组不同时间点血糖比NC组明显增加(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组尿蛋白(P0.01)随时间不断增加,差异有统计学意义(P0.01)。3)肾功能:(1)DN组不同时间点血清肌酐及尿素氮比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(P0.05)。3.免疫组化:(1)c-FLIP主要表达于肾小球和肾小管细胞中;DN组不同时间点c-FLIP的表达量比NC组明显减少,差异有统计学意义(P0.01);DN组c-FLIP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显著低于2W和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显著低于8W和12W,(P0.01)。(2)RIP1和RIP3也是主要表达于肾小球细胞中;DN组RIP1和RIP3不同时间点比NC组明显增加(P0.01);DN组以上两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和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和12w,差异有统计学意义(p0.01或p0.05)。(3)dn组c-flip蛋白表达量与rip1、rip3呈显著负相关(r=-0.933,p0.01;r=-0.954,p0.01),与尿蛋白、肾重指数、血清肌酐及尿素氮均也呈负相关(r=-0.945,p0.01;r=-0.922,p0.01;r=-0.543,p0.01;r=-0.494,p0.01)。4.免疫印迹:(1)dn组不同时间点c-flip的表达量比nc组明显减少,差异有统计学意义(p0.01);dn组c-flip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显著低于2w和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显著低于8w、12w,(p0.01)。(2)dn组rip1和rip3不同时间点比nc组明显增加(p0.01);dn组以上两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和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和12w,差异有统计学意义(p0.01)。(3)dn组c-flip蛋白表达量与rip1、rip3呈显著负相关(r=-0.985,p0.01;r=-0.986,p0.01),与尿蛋白、肾重指数、血清肌酐及尿素氮也呈负相关(r=-0.930,p0.01;r=-0.907,p0.01;r=-0.575,p0.01;r=-0.558,p0.01)。5.real-timepcr:(1)dn组不同时间点c-flipmrna相对表达量比nc组明显减少,差异有统计学意义(p0.01);dn组c-flip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显著低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显著低于8w和12w(p0.01)。(2)dn组rip1和rip3不同时间点比nc组明显增加(p0.01);dn组以上两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和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和12w,差异有统计学意义(p0.01)。(3)dn组c-flipmrna相对表达量与rip1、rip3呈显著负相关(r=-0.805,p0.01;r=-0.869,p0.01),与尿蛋白、肾重指数、血清肌酐及尿素氮也呈负相关(r=-0.877,p0.01;r=-0.895,p0.01;r=-0.643,p0.01;r=-0.499,p0.01)。结论:1.糖尿病大鼠肾组织中c-FLIP的表达量减少,并与糖尿病肾组织损伤有关;2.糖尿病大鼠肾组织中细胞坏死性凋亡标志物RIP1和RIP3表达增加,坏死性凋亡途径的激活可导致肾脏组织损伤;3.c-FLIP的表达量与RIP1和RIP3呈负相关,糖尿病肾病时,c-FLIP在肾脏中的低表达可能引起坏死性凋亡途径的激活,进而导致肾脏组织损伤。
[Abstract]:Objective: to construct diabetic rat model with streptozotocin (STZ) and explore the expression of Fas related dead domain protein like interleukins -1 beta converting enzyme inhibitor (c-FLIP) in renal tissue of diabetic nephropathy rats and the role of c-FLIP mediated apoptosis pathway in the pathogenesis of diabetic nephropathy. Methods: adult male adult SD rats Standardized feeding. The experimental group was divided into diabetic nephropathy model group (DN) and normal control group (NC), group DN was intraperitoneal injection of streptozotocin (STZ) 55mg/Kg, each group of 6 rats at 5 observation points at each time, each time point, the blood, urine specimen, serum creatinine, serum urea nitrogen and 24 hour urine protein were measured before death. Visceral paraffin section was used for common pathology; immunohistochemistry and immunoblotting (WB) were used to determine the expression of c-FLIP, receptor interaction protein 1 (RIP1) and receptor interacting protein 3 (RIP3) in renal tissue. RT-PCR was used to determine the c-FLIP, RIP1 and RIP3 mRNA relative expression of renal tissue. The correlation between the indexes was analyzed. Results: 1. blood, urine index: 1) blood sugar: DN group was not The blood glucose at the same time point was significantly increased (P0.01).2) 24 hours urine protein: (1) the urine protein in the group DN was significantly higher than that in the NC group (P0.01). (2) the urinary protein (P0.01) in the DN group increased with time, and the difference was statistically significant (P0.01).3) renal function: (1) the serum creatinine and urea nitrogen in the DN group were significantly increased (2) group (2) There was no significant difference in serum creatinine and urea nitrogen (P0.05).2. renal pathology: (1) the kidney of group DN was white and swollen, and the glomerular hypertrophy, cell number increased, mesangial matrix increased, partial renal tubule dilatation and vacuolated degeneration. (2) the renal weight index of group DN was significantly higher than that in group NC (3) DN group kidney weight. The index increased with time, and the difference was statistically significant (P0.05).3. immunohistochemistry: (1) c-FLIP was mainly expressed in the glomeruli and renal tubule cells, and the expression of c-FLIP in group DN was significantly lower than that of NC group at different time points, and the difference was statistically significant (P0.01). The difference of c-FLIP expression at different time points in DN group was no statistical difference between 2W and 4W. 8W, 12W, 16W were significantly lower than 2W and 4W (P0.01), and there was no statistical difference between 8W and 12W, 16W was significantly lower than 8W and 12W, (P0.01). (2) The difference between 2W and 4W was not statistically significant, 8W, 12W, 16W were significantly higher in 2W and 4W, and the difference was statistically significant (P0.01). The difference between 8W and 12W was not statistically significant, and 16W was significantly higher than 8W and 12W. (3) 954, P0.01) was also negatively correlated with urine protein, renal weight index, serum creatinine and urea nitrogen (r=-0.945, P0.01; r=-0.922, P0.01; r=-0.543, P0.01; r=-0.494, P0.01).4. immunoblotting: (1) the expression of c-FLIP was significantly reduced at different time points in the DN group, and the difference was statistically significant (2). The difference between W and 4W was not statistically significant, 8W, 12W, 16W were significantly lower than 2W and 4W, and the difference was statistically significant (P0.01). The difference between 8W and 12W was not statistically significant, and 16W was significantly lower than 8W. (2) the different time points were significantly increased. There was no statistically significant difference in 8W, 12W and 16W, and there were significant differences in 2W and 4W (P0.01). There was no statistically significant difference between 8W and 12W, 16W significantly higher than 8W and 12W, the difference was statistically significant (P0.01). (3) there was a significant negative correlation between the expression of proteinuria and the urinary protein and renal heavy finger. The number of serum creatinine and urea nitrogen also showed negative correlation (r=-0.930, P0.01; r=-0.907, P0.01; r=-0.575, P0.01; r=-0.558, P0.01).5.real-timepcr: (1) DN group at different time points, the relative expression of c-flipmrna was significantly lower than that of the NC group, and the difference was statistically significant. 8W, 12W, and 16W were significantly lower 2W, 4W, and the difference was statistically significant (P0.01). The difference between 8W and 12W was not statistically significant, 16W was significantly lower than 8W and 12W (P0.01). (2) the difference of time points between the two groups was significantly higher than that of the 12W. 12W and 16W were significantly higher 2W and 4W, the difference was statistically significant (P0.01). There was no statistically significant difference between 8W and 12W, 16W was significantly higher than 8W and 12W, and the difference was statistically significant (P0.01). (3) the relative expression of the DN group was negatively correlated with the urinary protein, renal weight index, serum creatinine and urea nitrogen. It also showed negative correlation (r=-0.877, P0.01; r=-0.895, P0.01; r=-0.643, P0.01; r=-0.499, P0.01). Conclusion: 1. the expression of c-FLIP in renal tissue of diabetic rats is reduced, and it is related to diabetic renal tissue injury. 2. the expression of necrotic apoptosis marker in renal tissue of diabetic rats increases, and the activation of necrotic apoptosis pathway can lead to the activation of necrotic apoptosis pathway. The expression of 3.c-FLIP is negatively correlated with RIP1 and RIP3. In diabetic nephropathy, the low expression of c-FLIP in the kidney may cause the activation of necrotic apoptotic pathway, which may lead to renal tissue damage.
【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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