【摘要】：目的： 探索“代謝性記憶”對(duì)糖尿病腎病進(jìn)展的作用，進(jìn)一步完善糖尿病腎病的發(fā)病機(jī)制。 方法： 1．第一部分：（動(dòng)物實(shí)驗(yàn)）：成年雄性Wistar大鼠，血糖在正常范圍內(nèi)。將實(shí)驗(yàn)用大鼠隨機(jī)分成3組：正常對(duì)照組（NC組，n=15）、鏈脲佐菌素(streptozocin,STZ)誘導(dǎo)的DM模型組（DM組，n＝15）、DM+胰島素治療組（DI組，n＝15）。NC、DM、DI各組均喂以普通飼料。其中DI組為DM造模成功4周后開始每天予以胰島素頸背部皮下注射控制血糖，單只總用量為6-8IU/D，每日測(cè)尾靜脈血糖以便調(diào)整胰島素用量，將隨機(jī)血糖控制在11.1mmol/L以下；NC組及DM組則每天給予等量生理鹽水皮下注射，持續(xù)觀察8周。測(cè)定各實(shí)驗(yàn)組的血糖、血白蛋白、血肌酐、血尿素氮及胱抑素C濃度；PAS染色觀察腎組織的形態(tài)學(xué)改變，并應(yīng)用免疫組化方法觀察PAI-1、CTGF在腎臟中的表達(dá)。 2．第二部分：（細(xì)胞實(shí)驗(yàn)）：分為4組：(1)正常血糖組(NG)；(2)高血糖組（HG）；(3)高血糖轉(zhuǎn)正常血糖組(HG-NG)；(4)正常血糖組+甘露醇（NG+M）；應(yīng)用Realtime-qPCR方法檢測(cè)CTGF、PAI-1的表達(dá)。 結(jié)果： 生化檢查：與NC組相比，DM組及DI組24小時(shí)尿蛋白定量均顯著增加，DM、DI兩組之間無顯著差異，，DM組和DI組大鼠血漿白蛋白均明顯降低(P0.01)，DI組與DM組之間無顯著差異。DM組大鼠血糖較NC組明顯升高，且維持在較高水平上（P0.01），而DI組與DM組相比，血糖明顯受到控制（P0.01），接近于正常組水平。各組之間相比較，大鼠血肌酐、血尿素氮、胱抑素濃度無顯著性差異。 大鼠腎臟組織腎組織PAS及PAM染色顯示，與正常組相比，DM組和DI組表現(xiàn)為腎小球體積增大，系膜細(xì)胞輕度增生，系膜基質(zhì)彌漫增多，部分可見毛細(xì)血管基底膜增厚，腎間質(zhì)輕度水腫。而NC組則無明顯病理改變。 Realtime-qPCR結(jié)果表明：細(xì)胞培養(yǎng)的系膜細(xì)胞CTGF-mRNA水平：與NG和NG+M組比較，HG組和HG-NG組CTGF-mRNA水平均增加，且差異具有顯著性。HG組與HG-NG組比較，CTGF-mRNA水平表達(dá)略有增加，但差異無統(tǒng)計(jì)學(xué)意義（P＞0.05）。而NG-M組與NG組相比稍增加，差異無統(tǒng)計(jì)學(xué)意義，故可說明高血糖組并非因滲透壓原因影響CTGF的表達(dá)增加。PAI-1的mRNA表達(dá)水平在上述刺激環(huán)境下趨勢(shì)與CTGF表達(dá)大致相當(dāng)。 結(jié)論： 1、通過動(dòng)物實(shí)驗(yàn)表明：糖尿病大鼠應(yīng)用胰島素治療，將血糖控制至正常范圍后，24小時(shí)尿蛋白增加的現(xiàn)象未得到明顯改善；腎組織中纖維化因子CTGF、PAI-1仍有高表達(dá)。 2、通過細(xì)胞實(shí)驗(yàn)表明：糖尿病大鼠系膜細(xì)胞高糖轉(zhuǎn)正常血糖處理后，纖維化因子CTGF、PAI-1的mRNA水平未因血糖下降而降低。 3、上述結(jié)果支持高血糖得到控制后，腎臟組織及細(xì)胞中有“代謝性記憶”發(fā)生，能進(jìn)一步促進(jìn)糖尿病腎病的發(fā)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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R587.2;R692

【共引文獻(xiàn)】

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