“代谢性记忆”对糖尿病肾病进展的作用

发布时间：2018-09-14 16:38

【摘要】：目的：探索“代谢性记忆”对糖尿病肾病进展的作用，进一步完善糖尿病肾病的发病机制。方法： 1．第一部分：（动物实验）：成年雄性Wistar大鼠，血糖在正常范围内。将实验用大鼠随机分成3组：正常对照组（NC组，n=15）、链脲佐菌素(streptozocin,STZ)诱导的DM模型组（DM组，n＝15）、DM+胰岛素治疗组（DI组，n＝15）。NC、DM、DI各组均喂以普通饲料。其中DI组为DM造模成功4周后开始每天予以胰岛素颈背部皮下注射控制血糖，单只总用量为6-8IU/D，每日测尾静脉血糖以便调整胰岛素用量，将随机血糖控制在11.1mmol/L以下；NC组及DM组则每天给予等量生理盐水皮下注射，持续观察8周。测定各实验组的血糖、血白蛋白、血肌酐、血尿素氮及胱抑素C浓度；PAS染色观察肾组织的形态学改变，并应用免疫组化方法观察PAI-1、CTGF在肾脏中的表达。 2．第二部分：（细胞实验）：分为4组：(1)正常血糖组(NG)；(2)高血糖组（HG）；(3)高血糖转正常血糖组(HG-NG)；(4)正常血糖组+甘露醇（NG+M）；应用Realtime-qPCR方法检测CTGF、PAI-1的表达。结果：生化检查：与NC组相比，DM组及DI组24小时尿蛋白定量均显著增加，DM、DI两组之间无显著差异，，DM组和DI组大鼠血浆白蛋白均明显降低(P0.01)，DI组与DM组之间无显著差异。DM组大鼠血糖较NC组明显升高，且维持在较高水平上（P0.01），而DI组与DM组相比，血糖明显受到控制（P0.01），接近于正常组水平。各组之间相比较，大鼠血肌酐、血尿素氮、胱抑素浓度无显著性差异。大鼠肾脏组织肾组织PAS及PAM染色显示，与正常组相比，DM组和DI组表现为肾小球体积增大，系膜细胞轻度增生，系膜基质弥漫增多，部分可见毛细血管基底膜增厚，肾间质轻度水肿。而NC组则无明显病理改变。 Realtime-qPCR结果表明：细胞培养的系膜细胞CTGF-mRNA水平：与NG和NG+M组比较，HG组和HG-NG组CTGF-mRNA水平均增加，且差异具有显著性。HG组与HG-NG组比较，CTGF-mRNA水平表达略有增加，但差异无统计学意义（P＞0.05）。而NG-M组与NG组相比稍增加，差异无统计学意义，故可说明高血糖组并非因渗透压原因影响CTGF的表达增加。PAI-1的mRNA表达水平在上述刺激环境下趋势与CTGF表达大致相当。结论： 1、通过动物实验表明：糖尿病大鼠应用胰岛素治疗，将血糖控制至正常范围后，24小时尿蛋白增加的现象未得到明显改善；肾组织中纤维化因子CTGF、PAI-1仍有高表达。 2、通过细胞实验表明：糖尿病大鼠系膜细胞高糖转正常血糖处理后，纤维化因子CTGF、PAI-1的mRNA水平未因血糖下降而降低。 3、上述结果支持高血糖得到控制后，肾脏组织及细胞中有“代谢性记忆”发生，能进一步促进糖尿病肾病的发展。
[Abstract]:Objective: to explore the effect of metabolic memory on the progression of diabetic nephropathy and improve the pathogenesis of diabetic nephropathy. Methods: 1. Part 1: (animal experiment): adult male Wistar rats, blood glucose is within normal range. The rats were randomly divided into three groups: normal control group (NC group), streptozotocin (streptozocin,STZ) -induced DM model group (DM group) and DM insulin therapy group (DI group). In DI group, the blood glucose was controlled by subcutaneous injection of insulin on the neck and back of the neck and back every day after the successful establishment of DM for 4 weeks. The total dosage was 6-8 IUU / D. The blood glucose of tail vein was measured daily in order to adjust the dosage of insulin, and the random blood glucose was controlled below 11.1mmol/L. NC group and DM group were subcutaneously injected with the same amount of saline daily for 8 weeks. Blood glucose, serum albumin, serum creatinine, blood urea nitrogen and cystatin C were detected by pas staining. The expression of PAI-1,CTGF in kidney was observed by immunohistochemical method. 2. The second part: (cell experiment): divided into 4 groups: (1) normal blood glucose group (NG); (2) hyperglycemia group (HG); (3) hyperglycemia group (HG-NG); (4) mannitol (NG M); was used to detect the expression of CTGF,PAI-1 by Realtime-qPCR in normal blood glucose group. Results: biochemical examination: compared with NC group and DI group, there was no significant difference between DM group and DI group in 24 hour urinary protein quantification. There was no significant difference between DM group and DI group. The plasma albumin in DM group and DI group were significantly decreased (P0.01). There was no significant difference. The blood glucose in DM group was significantly higher than that in NC group. And maintained at a high level (P0.01), but compared with the DM group, the blood glucose in DI group was significantly controlled (P0.01), close to the level of normal group. There was no significant difference in serum creatinine, blood urea nitrogen and cystatin concentration among the groups. PAS and PAM staining showed that the glomerular volume, Mesangial cell proliferation, Mesangial matrix diffuse increase and capillary basement membrane thickening were observed in DM and DI groups compared with the normal group. Mild renal interstitial edema. The results of Realtime-qPCR showed that the CTGF-mRNA level of Mesangial cells cultured in NC group was higher than that in NG and NG M group, and the CTGF-mRNA level in HG group and HG-NG group was higher than that in NG and NG M group. The expression of CTGF-mRNA in HG group was slightly higher than that in HG-NG group, but there was no significant difference between HG group and HG-NG group (P > 0. 05). However, there was no significant difference between NG-M group and NG group, which indicated that the mRNA expression of PAI-1 in hyperglycemia group was not affected by osmotic pressure. The trend of mRNA expression of PAI-1 was similar to that of CTGF expression in the above stimulated environment. Conclusion: 1. The results of animal experiments showed that the increase of urinary protein at 24 hours after insulin therapy was not significantly improved in diabetic rats. There is still high expression of fibrosis factor CTGF,PAI-1 in renal tissue. 2. The results of cell experiment showed that the mesangial cells of diabetic rats were treated with hyperglycemia and normal blood glucose. The level of mRNA of fibrosis factor CTGF,PAI-1 was not decreased because of the decrease of blood glucose. 3 the above results support that there is "metabolic memory" in renal tissue and cells after hyperglycemia is controlled, which can further promote the development of diabetic nephropathy.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692

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