本文選題：糖尿病腎病 + 壞死性凋亡　； 參考：《遵義醫學院》2017年碩士論文

【摘要】：目的:使用鏈脲佐菌素(STZ)構建糖尿病大鼠模型,探討細胞型Fas相關死亡域蛋白樣白介素-1β轉換酶抑制蛋白(c-FLIP)在糖尿病腎病大鼠腎組織中的表達以及c-FLIP介導壞死性凋亡通路在糖尿病腎病發病機制中的作用。方法:雄性成年SD大鼠,標準化喂養。實驗分為糖尿病腎病模型組(DN)和正常對照組(NC),DN組采用鏈脲菌素(STZ)55mg/Kg腹腔注射造模,每組2、4、8、12、16周5個觀察點,每個時點6只大鼠。處死前采取血液、尿液標本,檢測血糖、血清肌酐、血清尿素氮和24小時尿蛋白定量;腎臟石蠟切片作普通病理;免疫組化、免疫印跡(WB)測定腎組織中c-FLIP、受體交互作用蛋白1(RIP1)和受體交互作用蛋白3(RIP3)表達,RT-PCR測定腎組織的c-FLIP、RIP1及RIP3 mRNA相對表達量。并分析各指標之間的相關性。結果:1.血、尿指標:1)血糖:DN組不同時間點血糖比NC組明顯增加(P0.01)。2)24小時尿蛋白:(1)DN組不同時間點尿蛋白比NC組明顯增加(P0.01)。(2)DN組尿蛋白(P0.01)隨時間不斷增加,差異有統計學意義(P0.01)。3)腎功能:(1)DN組不同時間點血清肌酐及尿素氮比NC組明顯增加(P0.01)。(2)DN組血清肌酐及尿素氮差異均無統計學意義(P0.05)。2.腎臟病理:(1)DN組大鼠腎臟發白、腫大,可見不同程度的腎小球肥大、細胞數增多、系膜基質增多、部分腎小管擴張及空泡樣變性。(2)DN組不同時間點腎重指數比NC組明顯增加(P0.01)。(3)DN組腎重指數隨時間不斷增加,差異有統計學意義(P0.05)。3.免疫組化:(1)c-FLIP主要表達于腎小球和腎小管細胞中;DN組不同時間點c-FLIP的表達量比NC組明顯減少,差異有統計學意義(P0.01);DN組c-FLIP表達量不同時間點比較,均為2W與4W相比差異無統計學意義,8W、12W、16W顯著低于2W和4W,差異有統計學意義(P0.01),8W與12W相比差異無統計學意義,16W顯著低于8W和12W,(P0.01)。(2)RIP1和RIP3也是主要表達于腎小球細胞中;DN組RIP1和RIP3不同時間點比NC組明顯增加(P0.01);DN組以上兩個指標表達量不同時間點比較,均為2W與4W相比差異無統計學意義,8W、12W、16W顯著高2W和4W,差異有統計學意義(p0.01),8w與12w相比差異無統計學意義,16w顯著高于8w和12w,差異有統計學意義(p0.01或p0.05)。(3)dn組c-flip蛋白表達量與rip1、rip3呈顯著負相關(r=-0.933,p0.01;r=-0.954,p0.01),與尿蛋白、腎重指數、血清肌酐及尿素氮均也呈負相關(r=-0.945,p0.01;r=-0.922,p0.01;r=-0.543,p0.01;r=-0.494,p0.01)。4.免疫印跡:(1)dn組不同時間點c-flip的表達量比nc組明顯減少,差異有統計學意義(p0.01);dn組c-flip表達量不同時間點比較,均為2w與4w相比差異無統計學意義,8w、12w、16w顯著低于2w和4w,差異有統計學意義(p0.01),8w與12w相比差異無統計學意義,16w顯著低于8w、12w,(p0.01)。(2)dn組rip1和rip3不同時間點比nc組明顯增加(p0.01);dn組以上兩個指標表達量不同時間點比較,均為2w與4w相比差異無統計學意義,8w、12w、16w顯著高2w和4w,差異有統計學意義(p0.01),8w與12w相比差異無統計學意義,16w顯著高于8w和12w,差異有統計學意義(p0.01)。(3)dn組c-flip蛋白表達量與rip1、rip3呈顯著負相關(r=-0.985,p0.01;r=-0.986,p0.01),與尿蛋白、腎重指數、血清肌酐及尿素氮也呈負相關(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組不同時間點c-flipmrna相對表達量比nc組明顯減少,差異有統計學意義(p0.01);dn組c-flip表達量不同時間點比較,均為2w與4w相比差異無統計學意義,8w、12w、16w顯著低2w、4w,差異有統計學意義(p0.01),8w與12w相比差異無統計學意義,16w顯著低于8w和12w(p0.01)。(2)dn組rip1和rip3不同時間點比nc組明顯增加(p0.01);dn組以上兩個指標表達量不同時間點比較,均為2w與4w相比差異無統計學意義,8w、12w、16w顯著高2w和4w,差異有統計學意義(p0.01),8w與12w相比差異無統計學意義,16w顯著高于8w和12w,差異有統計學意義(p0.01)。(3)dn組c-flipmrna相對表達量與rip1、rip3呈顯著負相關(r=-0.805,p0.01;r=-0.869,p0.01),與尿蛋白、腎重指數、血清肌酐及尿素氮也呈負相關(r=-0.877,p0.01;r=-0.895,p0.01;r=-0.643,p0.01;r=-0.499,p0.01)。結論:1.糖尿病大鼠腎組織中c-FLIP的表達量減少,并與糖尿病腎組織損傷有關;2.糖尿病大鼠腎組織中細胞壞死性凋亡標志物RIP1和RIP3表達增加,壞死性凋亡途徑的激活可導致腎臟組織損傷;3.c-FLIP的表達量與RIP1和RIP3呈負相關,糖尿病腎病時,c-FLIP在腎臟中的低表達可能引起壞死性凋亡途徑的激活,進而導致腎臟組織損傷。
[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.
【學位授予單位】：遵義醫學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R587.2;R692.9

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