發(fā)布時(shí)間：2018-05-24 20:40

  本文選題：骨形態(tài)蛋白2 + 糖尿病腎病�。� 參考：《西南醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:血管鈣化是糖尿病患者并發(fā)心血管疾病的主要表現(xiàn)之一,也是糖尿病患者死亡的重要危險(xiǎn)因素。糖尿病腎病(Diabetic nephropathy,DN)作為糖尿病的嚴(yán)重并發(fā)癥之一,其發(fā)生發(fā)展和糖尿病血管損傷緊密相關(guān)。已有研究表明,糖尿病患者比非糖尿病患者更易發(fā)生動(dòng)脈血管鈣化,而DN患者的鈣化程度更為嚴(yán)重及普遍。目前對(duì)DN血管鈣化的研究大多集中在外周動(dòng)脈及冠狀動(dòng)脈,而對(duì)腎動(dòng)脈血管的改變知之甚少。本研究通過觀察糖尿病腎病基礎(chǔ)上給予增強(qiáng)血管鈣化的因素后,腎動(dòng)脈鈣化程度與腎功能進(jìn)展的關(guān)系,同時(shí)觀察糖尿病腎病大鼠腎動(dòng)脈上BMP2及其下游因子Smad1,Runx2和Osterix的基因及蛋白變化情況,探討糖尿病腎病時(shí)腎動(dòng)脈鈣化嚴(yán)重程度與糖尿病腎病進(jìn)展的關(guān)系,及BMP2/Smad1/Runx2/0sterix信號(hào)通路激活在糖尿病腎病大鼠腎動(dòng)脈鈣化的作用。方法:將64只SD大鼠隨機(jī)分為對(duì)照組(CON組,18只))、糖尿病腎病組(DN組,22只)、糖尿病腎病合并血管鈣化組(DN+VDN組,24只),DN組及DN+VDN組高脂飼料喂養(yǎng)4周后,予以單次腹腔注射1%鏈脲佐菌素(STZ)溶液35mg/kg誘導(dǎo)糖尿病,CON組注射同等體積的0.1mmol/L檸檬酸鹽緩沖液。72小時(shí)后測(cè)血糖,連續(xù)三天血糖≥16.7 mmol/L為糖尿病成模。2周后,24 h尿蛋白量大于30 mg,尿量原尿量50%者為DN模型制備成功。DN成模第二天,DN+VDN組以維生素D3(300 000U/kg)肌肉注射,將尼古丁(25 mg/kg)完全溶解于花生油中給DN+VDN組大鼠灌胃,9 h后再灌胃一次,其余兩組予等量花生油灌胃及生理鹽水肌注。8、12和16周時(shí)收集各組大鼠尿液檢測(cè)24小時(shí)尿白蛋白(24-h Upro);心臟采血檢測(cè)血清尿素氮(BUN)、肌酐(Scr)、空腹血糖、胱抑素C(CysC)。大鼠腎臟組織病理檢查:清潔取腎,石蠟包埋后行HE染色,觀察腎小球和腎小管間質(zhì)的基本病理改變情況。摘取雙側(cè)腎動(dòng)脈用生理鹽水沖洗后,Von Kossa染色觀察大鼠腎動(dòng)脈鈣鹽沉積,并用鈣離子試劑盒檢測(cè)腎動(dòng)脈組織鈣含量。免疫熒光雙染檢測(cè)各組大鼠腎動(dòng)脈組織BMP2/α-SMA和Runx2/α-SMA蛋白表達(dá)變化。免疫組化檢測(cè)大鼠腎動(dòng)脈組織BMP2、Smad1、Runx2及Osterix蛋白的表達(dá)。實(shí)時(shí)熒光定量(RT-PCR)檢測(cè)各組大鼠腎動(dòng)脈組織BMP2和Runx2的基因表達(dá)。結(jié)果:1、大鼠一般情況①體重:與CON組比較,模型組體重明顯減輕,但DN組及DN+VDN組各時(shí)間點(diǎn)體重?zé)o明顯統(tǒng)計(jì)學(xué)差異。②血糖:CON組血糖在正常水平范圍波動(dòng),DN組及DN+VDN組大鼠血糖值顯著高于同時(shí)間點(diǎn)CON組,且兩模型組間血糖無統(tǒng)計(jì)學(xué)差異。③血清尿素氮(BUN)、肌酐(Scr):與CON組相比,DN組及DN+VDN組大鼠BUN值于實(shí)驗(yàn)8周后明顯升高,12周后DN組和DN+VDN組血Scr亦顯著升高(P0.05),而DN組與DN+VDN組間BUN、Scr值均無明顯統(tǒng)計(jì)學(xué)差異。④胱抑素C(CysC):實(shí)驗(yàn)期間,DN組大鼠血清CysC水平明顯高于同時(shí)間點(diǎn)CON組,低于DN+VDN組(P0.05)。⑤24小時(shí)尿白蛋白(24-h Upro):8周后DN組及DN+VDN組大鼠24-h Upro較CON組開始上升,隨時(shí)間的延長(zhǎng)進(jìn)行性加重,并于16周末DN+VDN組大鼠的24-h Upro較同期DN組明顯升高(P0.05)。2、大鼠腎動(dòng)脈鈣化的特征:①Von Kossa染色:CON組大鼠腎動(dòng)脈組織各時(shí)間點(diǎn)均未見明顯異常,DN組及DN+VDN組大鼠8周末時(shí)即可見血管內(nèi)膜及中膜彈性纖維間大量黑色顆粒,且隨著時(shí)間延長(zhǎng),沉積的黑色顆粒逐漸增多聚集成團(tuán)。②鈣含量測(cè)定:隨時(shí)間的進(jìn)展,三組大鼠鈣含量均逐漸增加,且DN+VDN組大鼠腎動(dòng)脈組織各時(shí)間點(diǎn)鈣水平較DN組顯著升高,CON組大鼠較同期DN組明顯下降(P0.05)。3、免疫熒光檢測(cè)BMP2/α-SMA和Runx2/α-SMA的表達(dá):CON組大鼠腎動(dòng)脈組織中可見BMP2及Runx2均少量表達(dá)且各時(shí)間點(diǎn)無明顯變化;與同期CON組大鼠相比,DN組和DN + VDN組大鼠腎動(dòng)脈組織BMP2及Runx2表達(dá)的綠色熒光強(qiáng)度相對(duì)較強(qiáng),而α-SMA表達(dá)的紅色熒光相對(duì)較弱;與DN組相比,DN +VDN組大鼠腎動(dòng)脈血管中可見BMP2及Runx2蛋白的表達(dá)較同期DN組增加,α-SMA的表達(dá)減少;此外,隨時(shí)間的推移,DN和DN + VDN組大鼠α-SMA的熒光強(qiáng)度也隨BMP2或Runx2蛋白表達(dá)的增強(qiáng)而減弱。4、BMP2/Smad1/Runx2/Osterix信號(hào)蛋白免疫組化表達(dá):CON組腎動(dòng)脈組織中BMP2、Smad1、Runx2及Osterix在血管中膜均有散在微量表達(dá)。DN組大鼠腎動(dòng)脈組織中可見BMP2、Smad1、Runx2和Osterix蛋白在實(shí)驗(yàn)8周末時(shí)表達(dá)即開始明顯增加,陽性著色深,胞質(zhì)呈棕黃色,主要分布于動(dòng)脈血管中膜平滑肌細(xì)胞中,隨時(shí)間的進(jìn)展,表達(dá)逐漸增強(qiáng)。與同期DN組大鼠相比,可見DN+VDN組大鼠腎動(dòng)脈組織BMP2/Smad1/Runx2/Osterix信號(hào)通路的表達(dá)明顯升高。5、腎動(dòng)脈組織BMP2及Runx2 mRNA的表達(dá):RT-PCR結(jié)果示,對(duì)照組大鼠腎動(dòng)脈組織BMP2、Runx2 mRNA僅有微量表達(dá),且各時(shí)間點(diǎn)無統(tǒng)計(jì)學(xué)差異;模型組大鼠腎動(dòng)脈組織自實(shí)驗(yàn)第8周末起,BMP2和Runx2 mRNA水平開始增加,16周末時(shí)BMP2及Runx2 mRNA的表達(dá)量達(dá)最高值,此外,與DN組相比,DN+VDN組同期大鼠表現(xiàn)出更高的BMP2和Runx2 mRNA水平(P0.05)。6、腎臟HE染色顯示:CON組大鼠各時(shí)間點(diǎn)未見明顯異常;8周末時(shí)DN組大鼠可見腎小球體積增大,基底膜輕度增厚,系膜區(qū)增寬,腎小管上皮細(xì)胞偶見空泡樣或顆粒狀變性,隨時(shí)間的延長(zhǎng),12周末時(shí)可見基底膜增厚更明顯,偶見腎小動(dòng)脈壁玻璃樣變性,16周末時(shí)腎組織損傷進(jìn)一步加重,基底膜重度增厚,并可見節(jié)段性硬化;與DN組相比,DN+VDN組同期大鼠上述病理改變明顯加重。結(jié)論:1、DN在較早階段時(shí)即已存在腎動(dòng)脈等大血管的鈣化,而在鈣化血管中存在BMP2/Smad1/Runx2/Osterix信號(hào)通路的激活;2、腎動(dòng)脈鈣化的發(fā)生與嚴(yán)重程度與DN進(jìn)展密切相關(guān);3、BMP2/Smad1/Runx2/Osterix信號(hào)通路是DN腎動(dòng)脈血管鈣化重要的調(diào)節(jié)因素。
[Abstract]:Objective: vascular calcification is one of the major manifestations of cardiovascular disease in diabetic patients and an important risk factor for the death of diabetic patients. Diabetic nephropathy (DN) is one of the serious complications of diabetes. The development of diabetes mellitus is closely related to diabetic vascular injury. Non diabetic patients are more likely to have arterial calcification, and the degree of calcification in DN patients is more serious and widespread. Most of the studies on vascular calcification in DN are concentrated in the peripheral arteries and coronary arteries, but little is known about the changes in the renal artery. The relationship between the degree of renal artery calcification and the progression of renal function, and the changes in the gene and protein of BMP2 and its downstream factors Smad1, Runx2 and Osterix on the renal artery of diabetic nephropathy rats, and to explore the relationship between the severity of renal artery calcification and the progression of diabetic nephropathy and the activation of BMP2/Smad1/Runx2/0sterix signaling pathway in diabetic nephropathy. Methods: the role of renal artery calcification in diabetic nephropathy rats. Methods: 64 SD rats were randomly divided into control group (group CON, 18), diabetic nephropathy group (group DN, 22), diabetic nephropathy combined with vascular calcification group (group DN+VDN, 24), DN group and DN+VDN group high fat feed for 4 weeks, and single intraperitoneal injection of 1% streptozotocin (STZ) solution 35mg/kg Induced diabetes, group CON was injected with the same volume of 0.1mmol/L citrate buffer for.72 hours after.72 hours. After three days of blood glucose more than 16.7 mmol/L for.2 weeks, the protein amount of 24 h was greater than 30 mg, and the urine volume of primary urine was 50% of the DN model for second days. The DN+VDN group was injected with vitamin D3 (300 000U/kg). Nicotine (25 mg/kg) was completely dissolved in peanut oil to gavage to rats in group DN+VDN and reperfusion after 9 h. The rest two groups were given equal amount of peanut oil for gastric perfusion and.8,12 and 16 weeks of physiological saline injection. Urine albumin (24-h Upro) was collected in each group of rats, and serum urea nitrogen (BUN), creatinine (Scr), fasting blood glucose, cystine, and cystine were suppressed by heart blood sampling. C (CysC). Pathological examination of kidney tissue in rats: after cleaning the kidneys and after paraffin embedding, HE staining was performed to observe the basic pathological changes of the interstitium in the glomeruli and renal tubules. After the extraction of bilateral renal arteries, the calcium salt deposition in the renal arteries was observed by Von Kossa staining and the calcium content of the renal artery was detected by the calcium ion kit. The expression of BMP2/ alpha -SMA and Runx2/ alpha -SMA protein in renal artery tissue of rats was detected by double staining. The expression of BMP2, Smad1, Runx2 and Osterix protein in renal artery tissue of rats was detected by immunohistochemistry. The expression of BMP2 and Runx2 in renal artery tissues of rats in each group was detected by real-time fluorescence quantitative (RT-PCR). Results: 1, the general condition of rats: and C In group ON, the body weight of the model group was significantly reduced, but there was no significant difference in weight at all time points between the DN group and the DN+VDN group. 2. The blood sugar in the group CON was fluctuated in the normal level, and the blood sugar of the DN group and the DN+VDN group was significantly higher than that in the CON group at the same time, and there was no statistical difference between the two model groups. (3) the serum urea nitrogen (BUN) and creatinine (Scr): and C. Compared with group ON, the BUN value of rats in group DN and DN+VDN group increased significantly after 8 weeks, and the blood Scr in DN and DN+VDN groups increased significantly after 12 weeks (P0.05), but there was no significant difference between the DN group and DN+VDN group. (5) 24 hour urinary albumin (24-h Upro): after 8 weeks, 24-h Upro in group DN and group DN+VDN began to rise and increased with time, and the 24-h Upro of DN+VDN group rats increased significantly at the end of the 16 week than that in DN group (P0.05). There was no obvious abnormality. In group DN and group DN+VDN, a large number of black particles were seen between the intima and the middle membrane elastic fibers at the end of 8 weeks. And with the time prolonging, the black particles were gradually increased and aggregated. The calcium content was measured in the three groups of rats with the progress of time, and the renal artery tissues of the group DN+VDN rats were all increased. Compared with the DN group, the level of calcium was significantly higher in the CON group than that in the DN group (P0.05).3. The expression of BMP2/ alpha -SMA and Runx2/ alpha -SMA were detected by immunofluorescence. The expression of BMP2 and Runx2 in the renal artery tissues of the rats of the CON group was small and there was no obvious change at each time point. The green fluorescence intensity expressed in BMP2 and Runx2 was relatively strong, while the red fluorescence expressed by alpha -SMA was relatively weak. Compared with the DN group, the expression of BMP2 and Runx2 protein in the renal artery of DN +VDN group was increased and the expression of alpha -SMA decreased. Besides, the fluorescence intensity of alpha -SMA in DN and DN + groups also followed the time. The enhancement of the expression of MP2 or Runx2 protein weakened.4 and the expression of BMP2/Smad1/Runx2/Osterix signal protein: BMP2, Smad1, Runx2 and Osterix in the renal artery tissues of group CON were scattered in the microexpression of the vascular membrane, and the BMP2 was seen in the renal artery tissue of the rats in the.DN group, and the expression began to increase obviously at the end of the 8 week of the experiment. In addition, the positive staining was deep and the cytoplasm was brown and yellow, mainly distributed in the membrane smooth muscle cells in the arterial blood vessels. The expression gradually increased with the development of time. Compared with the DN group, the expression of BMP2/Smad1/Runx2/Osterix signaling pathway in the renal artery tissue of the DN+VDN group was obviously elevated, and the expression of BMP2 and Runx2 mRNA in the renal artery tissue: RT-PC R results showed that the renal artery tissue of the rats in the control group was BMP2 and Runx2 mRNA only micro expression, and there was no statistical difference at all time points. The level of BMP2 and Runx2 mRNA in the renal artery tissue of the model rats began to increase at the end of the eighth week, and the expression of BMP2 and Runx2 mRNA at the 16 weekend reached the highest value. In addition, the DN+VDN group was compared with the same period of the DN+VDN group. The higher BMP2 and Runx2 mRNA level (P0.05).6 and renal HE staining showed that there was no obvious abnormality in the time points of the CON group. At the 8 weekend, the glomerular volume increased, the basement membrane was slightly thickened, the mesangial area widened, and the renal tubular epithelial cells had vacuolated or granular degeneration. The basement membrane was seen at the 12 weekend and the basement membrane was visible with time. The thickening is more obvious. I see the hyaline degeneration of the renal arteriole wall, the renal tissue damage is further aggravated at the end of the 16 week, the basal membrane is thickened and the segmental sclerosis is seen. Compared with the group DN, the pathological changes in the DN+VDN group are obviously aggravated. Conclusion: 1, the calcification of the large vessels, such as the renal arteries, is present in the early stage, and in the calcified blood vessels, in the earlier stage, the DN is in the calcified vessel. The activation of BMP2/Smad1/Runx2/Osterix signaling pathway is found, 2, the occurrence and severity of renal artery calcification are closely related to the progress of DN, and 3, BMP2/Smad1/Runx2/Osterix signaling pathway is an important regulatory factor for the calcification of the DN renal artery.
【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R587.2

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9 于世家;任平;馬麗佳;李小娟;鄭曙琴;武明東;劉自力;薛麗輝;;糖尿病住院患者1344例回顧性分析[A];第六次中國(guó)中西醫(yī)結(jié)合糖尿病學(xué)術(shù)會(huì)議論文匯編[C];2002年

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