小管间质纤维化进程中C型利钠肽对基质金属蛋白酶及其组织抑制因子的调控效应

发布时间：2018-07-05 03:14

本文选题：C型利钠肽 + 基质金属蛋白酶　；参考：《安徽医科大学》2012年硕士论文

【摘要】：背景与目的小管间质纤维化(tubulointerstitial fibrosis, TIF)是各种原因导致的肾脏损害进展至终末期的共同途径。由于小管间质区域约占正常肾脏组织结构的90%左右，因此与肾小球损害相比，小管间质区域受损对肾脏功能的影响更为突出[1]。基质金属蛋白酶(matrix metalloproteinases, MMPs)及其组织抑制因子(tissue inhibitors ofmetalloproteinases, TIMPs)代谢紊乱参与TIF进程中细胞外基质(extracellularmatrix, ECM)重构及肾脏瘢痕形成。本研究旨在探讨小管间质纤维化(tubulointerstitial fibrosis, TIF)进程中C型利钠肽(C-type natriuretic peptide, CNP)对基质金属蛋白酶(matrix metalloproteinases, MMPs)及其组织抑制因子(tissueinhibitors of metalloproteinases, TIMPs)的调控效应。方法本实验分为两部分：（一）动物模型的建立：选用健康雄性8周龄Wistar大鼠48只。随机分为8组，每组6只，其中4组为单侧输尿管梗阻(unilateral ureteralobstruction, UUO)模型组，4组为假手术(sham-operated rats, SOR)组。消毒铺巾，以2%水合氯醛（0.2ml/kg）腹腔注射麻醉，，取左侧腹纵行切口。UUO模型组分离暴露左侧输尿管，在近肾门中上端1/3处以4-0丝线双重结扎后离断，逐层关腹；假手术组仅分离暴露左侧输尿管即逐层关腹。分别于术后3d、1m、2m和3m心脏穿刺处死大鼠，留取左侧肾脏。（二）实验室检验：血、尿液生化指标检测：所有大鼠处死前均禁食自由饮水置于代谢笼中收集24h尿液，双缩脲比色法进行尿蛋白定量。术中腹主动脉留取血液标本2ml，3000r/min离心5min分离血清，标准酶法测定血清总蛋白(total protein, TP)、白蛋白(albumin, Alb)、尿素氮(bloodurea nitrogen, BUN)和肌酐(creatinine, Cr)浓度。肾脏病理分析：肾脏组织离体后立即予以生理盐水灌洗，灭菌纱布吸干称重，计算肾重/体重比值(the ratio ofkidney weight to body weight, KW/BW)，测量肾皮质厚度。肾脏组织以4%多聚甲醛固定，石蜡包埋，4μm切片，HE染色。参照Park等[9]报道，在100倍光镜下随机选择20个不重叠视野，将TIF分为正常、轻度、中度和重度四级，分别赋值0~3分。荧光定量PCR、免疫组化和western blot检测单侧输尿管梗阻UUO大鼠术后1d、1m、2m和3m时肾脏CNP、MMP-2、MMP-9、TIMP-1、TIMP-2和IV型胶原(type IV collagen, Col-IV)mRNA和蛋白的表达情况。结果 UUO大鼠CNP、MMP-2和MMP-9mRNA表达虽然随TIF进展逐渐降低，但却显著高于相同观察时间点假手术SOR组(P0.05)；与此相反，UUO大鼠TIMP-1和TIMP-2mRNA表达随TIF进展逐渐升高，至术后2m和3m时显著高于SOR组(P0.05)。上述各观察指标在转录水平的表达趋势同样被免疫组化和westernblot实验所印证，其综合效应是促进Col-IV表达上调、推动纤维化进展。结论 TIF进程中肾脏组织CNP表达衰减可能是导致MMPs/TIMPs代谢紊乱和细胞外基质过度沉积的重要因素。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R726.9

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