本文選題：骨形態蛋白2 + 糖尿病腎病　； 參考：《西南醫科大學》2016年碩士論文

【摘要】：目的:血管鈣化是糖尿病患者并發心血管疾病的主要表現之一,也是糖尿病患者死亡的重要危險因素。糖尿病腎病(Diabetic nephropathy,DN)作為糖尿病的嚴重并發癥之一,其發生發展和糖尿病血管損傷緊密相關。已有研究表明,糖尿病患者比非糖尿病患者更易發生動脈血管鈣化,而DN患者的鈣化程度更為嚴重及普遍。目前對DN血管鈣化的研究大多集中在外周動脈及冠狀動脈,而對腎動脈血管的改變知之甚少。本研究通過觀察糖尿病腎病基礎上給予增強血管鈣化的因素后,腎動脈鈣化程度與腎功能進展的關系,同時觀察糖尿病腎病大鼠腎動脈上BMP2及其下游因子Smad1,Runx2和Osterix的基因及蛋白變化情況,探討糖尿病腎病時腎動脈鈣化嚴重程度與糖尿病腎病進展的關系,及BMP2/Smad1/Runx2/0sterix信號通路激活在糖尿病腎病大鼠腎動脈鈣化的作用。方法:將64只SD大鼠隨機分為對照組(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4周后,予以單次腹腔注射1%鏈脲佐菌素(STZ)溶液35mg/kg誘導糖尿病,CON組注射同等體積的0.1mmol/L檸檬酸鹽緩沖液。72小時后測血糖,連續三天血糖≥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周時收集各組大鼠尿液檢測24小時尿白蛋白(24-h Upro);心臟采血檢測血清尿素氮(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的基本病理改變情況。摘取雙側腎動脈用生理鹽水沖洗后,Von Kossa染色觀察大鼠腎動脈鈣鹽沉積,并用鈣離子試劑盒檢測腎動脈組織鈣含量。免疫熒光雙染檢測各組大鼠腎動脈組織BMP2/α-SMA和Runx2/α-SMA蛋白表達變化。免疫組化檢測大鼠腎動脈組織BMP2、Smad1、Runx2及Osterix蛋白的表達。實時熒光定量(RT-PCR)檢測各組大鼠腎動脈組織BMP2和Runx2的基因表達。結果: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組各時間點體重無明顯統計學差異。②血糖:CON組血糖在正常水平范圍波動,DN組及DN+VDN組大鼠血糖值顯著高于同時間點CON組,且兩模型組間血糖無統計學差異。③血清尿素氮(BUN)、肌酐(Scr):與CON組相比,DN組及DN+VDN組大鼠BUN值于實驗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值均無明顯統計學差異。④胱抑素C(CysC):實驗期間,DN組大鼠血清CysC水平明顯高于同時間點CON組,低于DN+VDN組(P0.05)。⑤24小時尿白蛋白(24-h Upro):8周后DN組及DN+VDN組大鼠24-h Upro較CON組開始上升,隨時間的延長進行性加重,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、大鼠腎動脈鈣化的特征:①Von Kossa染色:CON組大鼠腎動脈組織各時間點均未見明顯異常,DN組及DN+VDN組大鼠8周末時即可見血管內膜及中膜彈性纖維間大量黑色顆粒,且隨著時間延長,沉積的黑色顆粒逐漸增多聚集成團。②鈣含量測定:隨時間的進展,三組大鼠鈣含量均逐漸增加,且DN+VDN組大鼠腎動脈組織各時間點鈣水平較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BMP2/α-SMA和Runx2/α-SMA的表達:CON組大鼠腎動脈組織中可見BMP2及Runx2均少量表達且各時間點無明顯變化;與同期CON組大鼠相比,DN組和DN + VDN組大鼠腎動脈組織BMP2及Runx2表達的綠色熒光強度相對較強,而α-SMA表達的紅色熒光相對較弱;與DN組相比,DN +VDN組大鼠腎動脈血管中可見BMP2及Runx2蛋白的表達較同期DN組增加,α-SMA的表達減少;此外,隨時間的推移,DN和DN + VDN組大鼠α-SMA的熒光強度也隨BMP2或Runx2蛋白表達的增強而減弱。4、BMP2/Smad1/Runx2/Osterix信號蛋白免疫組化表達:CON組腎動脈組織中BMP2、Smad1、Runx2及Osterix在血管中膜均有散在微量表達。DN組大鼠腎動脈組織中可見BMP2、Smad1、Runx2和Osterix蛋白在實驗8周末時表達即開始明顯增加,陽性著色深,胞質呈棕黃色,主要分布于動脈血管中膜平滑肌細胞中,隨時間的進展,表達逐漸增強。與同期DN組大鼠相比,可見DN+VDN組大鼠腎動脈組織BMP2/Smad1/Runx2/Osterix信號通路的表達明顯升高。5、腎動脈組織BMP2及Runx2 mRNA的表達:RT-PCR結果示,對照組大鼠腎動脈組織BMP2、Runx2 mRNA僅有微量表達,且各時間點無統計學差異;模型組大鼠腎動脈組織自實驗第8周末起,BMP2和Runx2 mRNA水平開始增加,16周末時BMP2及Runx2 mRNA的表達量達最高值,此外,與DN組相比,DN+VDN組同期大鼠表現出更高的BMP2和Runx2 mRNA水平(P0.05)。6、腎臟HE染色顯示:CON組大鼠各時間點未見明顯異常;8周末時DN組大鼠可見腎小球體積增大,基底膜輕度增厚,系膜區增寬,腎小管上皮細胞偶見空泡樣或顆粒狀變性,隨時間的延長,12周末時可見基底膜增厚更明顯,偶見腎小動脈壁玻璃樣變性,16周末時腎組織損傷進一步加重,基底膜重度增厚,并可見節段性硬化;與DN組相比,DN+VDN組同期大鼠上述病理改變明顯加重。結論:1、DN在較早階段時即已存在腎動脈等大血管的鈣化,而在鈣化血管中存在BMP2/Smad1/Runx2/Osterix信號通路的激活;2、腎動脈鈣化的發生與嚴重程度與DN進展密切相關;3、BMP2/Smad1/Runx2/Osterix信號通路是DN腎動脈血管鈣化重要的調節因素。
[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.
【學位授予單位】：西南醫科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R587.2

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