本文選題：C型利鈉肽 + 基質金屬蛋白酶��； 參考：《安徽醫(yī)科大學》2012年碩士論文

【摘要】：背景與目的 小管間質纖維化(tubulointerstitial fibrosis, TIF)是各種原因導致的腎臟損害進展至終末期的共同途徑。由于小管間質區(qū)域約占正常腎臟組織結構的90%左右，因此與腎小球損害相比，小管間質區(qū)域受損對腎臟功能的影響更為突出[1]�；|金屬蛋白酶(matrix metalloproteinases, MMPs)及其組織抑制因子(tissue inhibitors ofmetalloproteinases, TIMPs)代謝紊亂參與TIF進程中細胞外基質(extracellularmatrix, ECM)重構及腎臟瘢痕形成。本研究旨在探討小管間質纖維化(tubulointerstitial fibrosis, TIF)進程中C型利鈉肽(C-type natriuretic peptide, CNP)對基質金屬蛋白酶(matrix metalloproteinases, MMPs)及其組織抑制因子(tissueinhibitors of metalloproteinases, TIMPs)的調控效應。 方法 本實驗分為兩部分：（一）動物模型的建立：選用健康雄性8周齡Wistar大鼠48只。隨機分為8組，每組6只，其中4組為單側輸尿管梗阻(unilateral ureteralobstruction, UUO)模型組，4組為假手術(sham-operated rats, SOR)組。消毒鋪巾，以2%水合氯醛（0.2ml/kg）腹腔注射麻醉，，取左側腹縱行切口。UUO模型組分離暴露左側輸尿管，在近腎門中上端1/3處以4-0絲線雙重結扎后離斷，逐層關腹；假手術組僅分離暴露左側輸尿管即逐層關腹。分別于術后3d、1m、2m和3m心臟穿刺處死大鼠，留取左側腎臟。（二）實驗室檢驗：血、尿液生化指標檢測：所有大鼠處死前均禁食自由飲水置于代謝籠中收集24h尿液，雙縮脲比色法進行尿蛋白定量。術中腹主動脈留取血液標本2ml，3000r/min離心5min分離血清，標準酶法測定血清總蛋白(total protein, TP)、白蛋白(albumin, Alb)、尿素氮(bloodurea nitrogen, BUN)和肌酐(creatinine, Cr)濃度。腎臟病理分析：腎臟組織離體后立即予以生理鹽水灌洗，滅菌紗布吸干稱重，計算腎重/體重比值(the ratio ofkidney weight to body weight, KW/BW)，測量腎皮質厚度。腎臟組織以4%多聚甲醛固定，石蠟包埋，4μm切片，HE染色。參照Park等[9]報道，在100倍光鏡下隨機選擇20個不重疊視野，將TIF分為正常、輕度、中度和重度四級，分別賦值0~3分。 熒光定量PCR、免疫組化和western blot檢測單側輸尿管梗阻UUO大鼠術后1d、1m、2m和3m時腎臟CNP、MMP-2、MMP-9、TIMP-1、TIMP-2和IV型膠原(type IV collagen, Col-IV)mRNA和蛋白的表達情況。 結果 UUO大鼠CNP、MMP-2和MMP-9mRNA表達雖然隨TIF進展逐漸降低，但卻顯著高于相同觀察時間點假手術SOR組(P0.05)；與此相反，UUO大鼠TIMP-1和TIMP-2mRNA表達隨TIF進展逐漸升高，至術后2m和3m時顯著高于SOR組(P0.05)。上述各觀察指標在轉錄水平的表達趨勢同樣被免疫組化和westernblot實驗所印證，其綜合效應是促進Col-IV表達上調、推動纖維化進展。 結論 TIF進程中腎臟組織CNP表達衰減可能是導致MMPs/TIMPs代謝紊亂和細胞外基質過度沉積的重要因素。
[Abstract]:Background and objective tubulointerstitial fibrosis (tubulointerstitial fibrosis, TIF) is a common pathway for the progression of renal damage to the end stage. Because tubulointerstitial area accounts for about 90% of the normal renal tissue structure, the damage of tubulointerstitial area has more prominent effect on renal function than glomerular damage [1]. The metabolic disorders of matrix metalloproteinases (matrix metalloproteinases,) and tissue inhibitor (tissue inhibitors ofmetalloproteinases, (TIMPs) are involved in extracellular matrix remodeling and renal scar formation. The aim of this study was to investigate the regulatory effects of C-type natriuretic peptides on matrix metalloproteinases (matrix metalloproteinases,) and tissue inhibitor (tissueinhibitors of metalloproteinases, (TIMPs) in the process of tubulointerstitial fibrosis (tubulointerstitial fibrosis,). Methods the experiment was divided into two parts: (1) Establishment of animal model: 48 healthy male 8 weeks old Wistar rats were selected. The rats were randomly divided into 8 groups with 6 rats in each group, among which 4 groups were sham-operated rates (sor) group with unilateral ureteral obstruction (unilateral ureteralobstruction, UUO) model. The left ureter was exposed in the left ventral longitudinal incision. The left ureter was separated and exposed in the proximal upper end of the renal hilus by double ligation of 4-0 silk thread, and then the left ureter was cut off layer by layer after double ligation of the proximal renal hilum. It was anesthetized by intraperitoneal injection of 2% chloral hydrate (0.2ml/kg), and the left ureter was closed layer by layer. In the sham operation group, the left ureter was isolated and the abdomen was closed layer by layer. The rats were killed 3 days after operation by cardiac puncture of 1 m and 3 m respectively, and left kidney was taken. (2) Laboratory test: biochemical indexes of blood and urine were detected: all rats were free drinking water before death and collected 24 hours urine in metabolic cage, and the urine protein was measured by biuret colorimetry. Blood samples were collected from abdominal aorta by centrifugation with 5min at 2 ml / r / min. Serum total protein (total protein, TP), albumin (albumin, Alb), urea nitrogen (bloodurea nitrogen, bun) and creatinine (Cr) were determined by standard enzyme method. Renal pathology: the kidney tissue was perfused with normal saline immediately after being isolated, the sterilized gauze was weighed by suction, the kidney weight / body weight ratio (the ratio ofkidney weight to body weight, KW / BW) was calculated, and the thickness of renal cortex was measured. Renal tissue was fixed with 4% paraformaldehyde and paraffin-embedded 4 渭 m sections were stained with HE. Referring to Park et al. [9], 20 unoverlapped visual fields were randomly selected under 100 times light microscope, and TIF was divided into normal, mild, moderate and severe levels, with a score of 0 ~ 3 respectively. The expression of TIMP-2 and type IV collagen (Col-IV) mRNA and protein in the kidney of UUO rats with unilateral ureteral obstruction were detected by fluorescence quantitative PCR, immunohistochemistry and western blot. Results the expression of MMP-2 and MMP-9 mRNA decreased gradually with the progression of TIF in UUO rats, but they were significantly higher than those in SOR group at the same observation time point (P0.05), on the contrary, the expressions of TIMP-1 and TIMP-2 mRNA in UUO rats gradually increased with the progression of TIF. At 2 m and 3 m after operation, it was significantly higher than that in sor group (P 0.05). The expression trend of the above observed indexes at the transcriptional level was also confirmed by immunohistochemical and westernblot experiments. The comprehensive effect was to promote the up-regulation of Col-IV expression and promote the progression of fibrosis. Conclusion the decrease of CNP expression in renal tissue during TIF may be an important factor leading to the metabolic disorder of MMPs / TIMPs and the excessive deposition of extracellular matrix.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R726.9

【參考文獻】

相關期刊論文 前1條

1 王曉紅,歐和生,符民桂,李淑蓮,龐永政,唐朝樞,劉乃奎;脂質體攜載C型利鈉利尿肽對血管效應的影響[J];中國藥理學通報;1999年05期

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