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

【摘要】：背景與目的 小管間質(zhì)纖維化(tubulointerstitial fibrosis, TIF)是各種原因?qū)е碌哪I臟損害進(jìn)展至終末期的共同途徑。由于小管間質(zhì)區(qū)域約占正常腎臟組織結(jié)構(gòu)的90%左右，因此與腎小球損害相比，小管間質(zhì)區(qū)域受損對(duì)腎臟功能的影響更為突出[1]�；|(zhì)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(jìn)程中細(xì)胞外基質(zhì)(extracellularmatrix, ECM)重構(gòu)及腎臟瘢痕形成。本研究旨在探討小管間質(zhì)纖維化(tubulointerstitial fibrosis, TIF)進(jìn)程中C型利鈉肽(C-type natriuretic peptide, CNP)對(duì)基質(zhì)金屬蛋白酶(matrix metalloproteinases, MMPs)及其組織抑制因子(tissueinhibitors of metalloproteinases, TIMPs)的調(diào)控效應(yīng)。 方法 本實(shí)驗(yàn)分為兩部分：（一）動(dòng)物模型的建立：選用健康雄性8周齡Wistar大鼠48只。隨機(jī)分為8組，每組6只，其中4組為單側(cè)輸尿管梗阻(unilateral ureteralobstruction, UUO)模型組，4組為假手術(shù)(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(cè)腹縱行切口。UUO模型組分離暴露左側(cè)輸尿管，在近腎門(mén)中上端1/3處以4-0絲線雙重結(jié)扎后離斷，逐層關(guān)腹；假手術(shù)組僅分離暴露左側(cè)輸尿管即逐層關(guān)腹。分別于術(shù)后3d、1m、2m和3m心臟穿刺處死大鼠，留取左側(cè)腎臟。（二）實(shí)驗(yàn)室檢驗(yàn)：血、尿液生化指標(biāo)檢測(cè)：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(jìn)行尿蛋白定量。術(shù)中腹主動(dòng)脈留取血液標(biāo)本2ml，3000r/min離心5min分離血清，標(biāo)準(zhǔn)酶法測(cè)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(chēng)重，計(jì)算腎重/體重比值(the ratio ofkidney weight to body weight, KW/BW)，測(cè)量腎皮質(zhì)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(bào)道，在100倍光鏡下隨機(jī)選擇20個(gè)不重疊視野，將TIF分為正常、輕度、中度和重度四級(jí)，分別賦值0~3分。 熒光定量PCR、免疫組化和western blot檢測(cè)單側(cè)輸尿管梗阻UUO大鼠術(shù)后1d、1m、2m和3m時(shí)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(dá)情況。 結(jié)果 UUO大鼠CNP、MMP-2和MMP-9mRNA表達(dá)雖然隨TIF進(jìn)展逐漸降低，但卻顯著高于相同觀察時(shí)間點(diǎn)假手術(shù)SOR組(P0.05)；與此相反，UUO大鼠TIMP-1和TIMP-2mRNA表達(dá)隨TIF進(jìn)展逐漸升高，至術(shù)后2m和3m時(shí)顯著高于SOR組(P0.05)。上述各觀察指標(biāo)在轉(zhuǎn)錄水平的表達(dá)趨勢(shì)同樣被免疫組化和westernblot實(shí)驗(yàn)所印證，其綜合效應(yīng)是促進(jìn)Col-IV表達(dá)上調(diào)、推動(dòng)纖維化進(jìn)展。 結(jié)論 TIF進(jìn)程中腎臟組織CNP表達(dá)衰減可能是導(dǎo)致MMPs/TIMPs代謝紊亂和細(xì)胞外基質(zhì)過(guò)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(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R726.9

【參考文獻(xiàn)】

相關(guān)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(zhì)體攜載C型利鈉利尿肽對(duì)血管效應(yīng)的影響[J];中國(guó)藥理學(xué)通報(bào);1999年05期

本文編號(hào)：2098674