本文選題：慢性腎臟病 + 血管鈣化��； 參考：《西南醫(yī)科大學》2017年碩士論文

【摘要】：目的:慢性腎臟病(Chronic kidney disease,CKD)患者往往合并血管鈣化,而血管鈣化是其心血管疾病(Cardiovascular disease,CVD)發(fā)生率和死亡率增高的主要原因。血管鈣化的發(fā)生與諸多因素相關,近年來骨形態(tài)發(fā)生蛋白相關信號通路已成為血管鈣化發(fā)生機制的研究熱點,但其具體機制尚未完全闡明。本研究采用腺嘌呤灌胃聯(lián)合高磷飼料喂養(yǎng)大鼠,建立CKD血管鈣化大鼠模型,觀察骨形態(tài)發(fā)生蛋白相關信號通路,包括骨形態(tài)發(fā)生蛋白-2(Bone morphogenetic protein-2,BMP-2)、骨形態(tài)發(fā)生蛋白-4(bone morphogenetic protein-2,BMP-4)、以及受體(Bone morphogenetic protein receptor-IA,BMPR-IA)、抑制劑(Matrix Gla Protein,MGP)在大鼠主動脈上的表達及激活情況,探討其作用機制,為臨床上早期防治CKD血管鈣化,降低患者心血管疾病的發(fā)病率和死亡率提供策略。方法:(1)CKD血管鈣化大鼠模型制備:190-270g左右SPF(無特定病原體)雄性大鼠55只,隨機分為對照組(CON,n=20)和CKD組(CKD,n=35),適應性喂養(yǎng)10天后,CKD組給予2.5%腺嘌呤(220-250 mg/kg.d)定時灌胃,第1-4周每日一次,第5-8周隔日一次,聯(lián)合1.8%高磷大鼠飼料喂養(yǎng);對照組予以生理鹽水(10ml/kg)灌胃,普通大鼠飼料喂養(yǎng),所有大鼠均自由進食及飲水,造模時間共8周。(2)指標檢測:第2、4、6、8周末,分別從CKD組及對照組隨機選取6只和5只大鼠處死收集標本。處死前一天,代謝籠收集尿液,檢測24小時尿蛋白定量;2%戊巴比妥鈉30-60mg/kg腹腔注射麻醉,腹主動脈取血,全自動生化分析儀檢測血尿素氮(BUN)、血肌酐(Scr)、胱抑素C(Cysc)、血鈣(Ca2+)、血磷(P3-);酶聯(lián)免疫吸附法(ELISA)測定血清BMP-2、BMP-4含量;測量腎重/體重指數(shù);腹主動脈取血后,快速分離腎臟,置于10%福爾馬林液固定,蘇木精-伊紅(HE)染色法觀察腎臟病理改變;快速剝離主動脈,按需分為三部分,一部分用10%中性福爾馬林固定,行Von Kossa染色、茜素紅染色;免疫組化檢測主動脈BMP-2、BMP-4、BMPR-IA(ALK3)、MGP蛋白表達;另一部分,存于裝有1ml RNA保護液的EP管中,-20℃冰箱凍存,用于實時熒光定量PCR測定BMP-2、BMP-4 m RNA表達;其余部分于-80℃冰箱保存,用于主動脈鈣含量測定。結果:1、一般情況:(1)死亡數(shù):對照組大鼠全部存活;CKD組大鼠第2周出現(xiàn)死亡,死亡1只,實驗總過程共死亡9只。(2)大鼠體重、腎重、腎重/體重指數(shù):與對照組各時間點相比,CKD組大鼠體重明顯降低(P0.05);而腎重、腎重/體重指數(shù)均明顯增加(P0.05,P0.05)。2、24h尿蛋白定量:與對照組各時間點相比,CKD組大鼠24h尿蛋白定量明顯增加(P0.01),且CKD組大鼠隨時間進展24h尿蛋白定量逐漸增加(P0.01)。3、血清學指標:(1)BUN、Scr、Cysc:與對照組相比,CKD組大鼠各時間點血清BUN、Scr、Cysc水平明顯增高(P0.01,P0.01,P0.01)。(2)血清鈣、磷、鈣磷乘積:與對照組相比,CKD組大鼠各時間點血磷及鈣磷乘積水平明顯增加(P0.01,P0.01),血鈣水平自第4周出現(xiàn)明顯降低(P0.05)。4、ELISA法測定血清BMP-2、BMP-4含量:(1)BMP-2:與對照組相比,CKD組大鼠各時間點血清BMP-2水平明顯增加(P0.05)。此外,CKD組大鼠血清BMP-2隨時間進展逐漸增加(P0.05)。(2)BMP-4:與對照組相比較,CKD組大鼠血清BMP-4水平自第4周開始均較同時間點增高(P0.01)。此外,CKD組大鼠血清BMP-4水平第6周、第8周時較第4周降低(P0.01)。5、腎臟病理改變:(1)肉眼:對照組大鼠腎臟大小適中,顏色暗紅,表面光滑,質(zhì)地柔軟,皮髓質(zhì)分界清晰;CKD組大鼠腎臟呈“大白腎”改變,顏色灰白,表面不光滑,彈性差。(2)腎臟HE染色:對照組腎臟形態(tài)、結構無異常改變。CKD組自第2周起,出現(xiàn)腎小管擴張,可見小管內(nèi)棕黃色物質(zhì)沉積,隨時間進展,第4,6,8周逐漸出現(xiàn)腎小球囊腔擴張,間質(zhì)纖維化,腎小球部分萎縮,炎性細胞浸潤,血管減少。6、主動脈形態(tài)改變:對照組主動脈光滑,彈性好。CKD組自第4周開始主動脈逐漸彎曲、膨大,呈動脈瘤樣改變,彈性降低,第6,8周血管僵硬度明顯增加,可見鈣化結節(jié)形成。7、主動脈鈣染色:(1)Von Kossa染色:對照組大鼠各時間點主動脈形態(tài)均正常,未見黑色顆粒物沉積。CKD組主動脈自第4周開始出現(xiàn)黑色顆粒沉積,伴中膜平滑肌纖維斷裂,鈣化結節(jié)處尤其明顯,且隨時間進展黑色顆粒沉積逐漸加重。(2)茜素紅染色:對照組大鼠各個時間點主動脈形態(tài)均正常,未見橘紅色物質(zhì)沉積。CKD組主動脈自第4周開始逐漸出現(xiàn)中膜平滑肌橘紅色顆粒物沉積,伴平滑肌纖維斷裂,鈣化結節(jié)處沉積更明顯,且隨時間進展橘紅色顆粒物沉積逐漸加重。8、主動脈鈣含量:與對照組各時間點比較,CKD組大鼠主動脈鈣含量明顯增加(P0.01),且CKD組內(nèi)隨時間進展鈣含量逐漸增加(P0.01)。9、主動脈BMP-2、BMP-4、BMPR-IA(ALK3)、MGP免疫組化結果:對照組大鼠主動脈各時間點BMP-2、BMPR-IA、MGP蛋白在血管中膜平滑肌層幾乎無表達,BMP-4在各時間點存在少量表達。與對照組相比,CKD組大鼠第4周起可見主動脈上BMP-2、BMPR-IA、MGP蛋白表達開始明顯增加,胞質(zhì)呈棕黃色,主要分布于主動脈中膜平滑肌細胞層,且隨時間進展,其表達逐漸增強(P0.01);BMP-4蛋白自第4周表達明顯增加,第6周、第8周較第4周稍下降,但與對照組同時間點比較仍然明顯增高(P0.01)。10、實時熒光定量PCR法檢測主動脈BMP-2、BMP-4m RNA表達量:(1)BMP-2m RNA:對照組各時間點主動脈BMP-2m RNA的表達無明顯變化;與對照組各時間點相比較,CKD組主動脈BMP-2m RNA表達量明顯增加(P0.01)。且隨時間進展,CKD組主動脈BMP-2m RNA表達逐漸增加(P0.01)。(2)BMP-4m RNA:與對照組各時間點比較,CKD組主動脈BMP-4m RNA表達量自第4周后均明顯高于對照組(P0.01);第6周、8周較第4周降低(P0.01)。結論:(1)腺嘌呤灌胃聯(lián)合高磷飼料喂養(yǎng)大鼠,能夠快速建立慢性腎臟病血管鈣化動物模型。(2)慢性腎臟病大鼠血管鈣化早期即存在BMP-2、BMP-4及其受體BMPR-IA(ALK3)、抑制劑MGP的蛋白及基因表達明顯增高,說明骨形態(tài)發(fā)生蛋白相關信號通路的激活參與CKD血管鈣化的發(fā)生發(fā)展。(3)CKD血管鈣化大鼠血清BMP-2、BMP-4水平明顯增高,且與主動脈鈣化程度呈正相關,可能可作為CKD血管鈣化的血清標志物。(4)慢性腎臟病血管鈣化過程中,BMP-4的成骨活性及表達特異性可能弱于BMP-2。
[Abstract]:Objective: Patients with Chronic kidney disease (CKD) often merge with vascular calcification, and vascular calcification is the main cause of the increase in the incidence of Cardiovascular disease (CVD) and the increase in mortality. The occurrence of vascular calcification is related to many factors. In recent years, the signal pathway related to bone morphogenetic protein has become vascular calcium. In this study, the rat model of CKD vascular calcification was established by adenine gavage and high phosphorus diet. The bone morphogenetic protein -2 (Bone morphogenetic protein-2, BMP-2) and bone morphogenetic protein -4 (bone morphogenetic protein -4) were observed in this study. Bone morphogenetic protein-2, BMP-4), and the expression of the receptor (Bone morphogenetic protein receptor-IA, BMPR-IA), the expression and activation of the inhibitor (Matrix Gla Protein) on rat aorta, and explore the mechanism of its action to provide strategies for the early prevention and treatment of vascular calcification and the reduction of the incidence and mortality of cardiovascular disease in patients. Methods: (1) CKD vascular calcification rat model was prepared: 190-270g SPF (without specific pathogens) 55 male rats, randomly divided into control group (CON, n=20) and CKD group (CKD, n=35), adaptive feeding for 10 days, CKD group given 2.5% adenine (220-250 mg/kg.d) regularly gavage, the 1-4 week, the next day, the next day, combined with 1.8% high phosphorus rats Feed feeding, the control group was given the normal saline (10ml/kg) gavage, the normal rat feed was fed, all rats were free to eat and drink, and the model time was 8 weeks. (2) the index test: at the end of the week 2,4,6,8, 6 and 5 rats were randomly selected from the CKD group and the control group. The urine was collected on the day before the death, and the urine was collected for 24 hours. Urine protein quantitative; 2% pentobarbital sodium 30-60mg/kg intraperitoneal injection anesthesia, abdominal aorta blood extraction, full automatic biochemical analyzer to detect blood urea nitrogen (BUN), serum creatinine (Scr), Cystatin C (Cysc), blood calcium (Ca2+), blood phosphorus (P3-); enzyme linked immunosorbent assay (ELISA) determination of serum BMP-2, BMP-4 content; measurement of kidney weight / body mass index; abdominal aorta blood, rapid after the blood, rapid The kidneys were separated and placed in 10% formalin fluid, and hematoxylin eosin (HE) staining was used to observe the pathological changes of the kidney; the rapid stripping aorta was divided into three parts according to the need. Part of the 10% neutral formalin was fixed with 10% neutral formalin. Von Kossa staining, alizarin red staining, and immunohistochemical detection of aorta BMP-2, BMP-4, BMPR-IA (ALK3), MGP protein expression; and the other part, Stored in EP tube with 1ml RNA protection liquid, cryopreservation at -20 C refrigerator, used for real-time quantitative PCR determination of BMP-2, BMP-4 m RNA expression, and the rest in the refrigerator of -80 C for the determination of calcium content in the aorta. Results: 1, general conditions: (1) the number of deaths in the control group was all alive, CKD group died in second weeks and 1 died in the total experiment. 9 rats were killed. (2) weight, kidney weight, kidney weight / body mass index of rats: compared with the control group, the weight of rats in group CKD decreased significantly (P0.05), while the kidney weight, kidney weight / body mass index increased significantly (P0.05, P0.05).2,24h urine protein quantitative: compared with the control group, the quantitative increase of 24h urine protein in the CKD group was significantly increased (P0.01), and CKD group. 24h urine protein quantitative increase (P0.01).3, serological index: (1) BUN, Scr, Cysc: compared with the control group, the serum BUN, Scr, Cysc levels in the CKD group were significantly higher (P0.01, P0.01, and Cysc:) in the CKD group. (2) the serum calcium, phosphorus, calcium and phosphorus product: compared with the control group, the level of phosphorus and calcium phosphorus in the rats at all time points was obvious Increase (P0.01, P0.01), blood calcium level appeared significantly decreased from fourth weeks (P0.05).4, ELISA method was used to determine serum BMP-2, BMP-4 content: (1) BMP-2: and control group compared with the control group, the serum BMP-2 level of rats at each time point increased significantly (P0.05). Besides, the CKD group rat serum progressively increased with time. (2) compared with the control group, BMP-2: The serum BMP-4 level of rats increased from fourth weeks to the same time point (P0.01). In addition, the serum level of BMP-4 in CKD group was sixth weeks, eighth weeks decreased (P0.01).5 and renal pathological changes: (1) naked eye: the kidney of the control group was moderate in size, dark red, smooth, soft, and clear in the demarcation of the skin and medulla; the kidney of the group of CKD group was " The color of the kidney was changed, the color was gray, the surface was not smooth, and the elasticity was poor. (2) the kidney HE staining: the renal morphology and structure of the control group had no abnormal changes in the.CKD group. The renal tubular dilatation appeared from second weeks, and the brown yellow substance was deposited in the tubule. The glomerular cysts dilation, interstitial fibrosis and partial atrophy of glomeruli gradually appeared on week 4,6,8. Inflammatory cell infiltration, vascular reduction.6, aorta morphologic changes: the aorta was smooth in the control group, and the aorta was smooth in the control group. The aorta was gradually curved and expanded from fourth weeks. The aneurysm like changes, the elasticity decreased, the vascular stiffness increased significantly at the end of the 6,8 week, the calcified nodules formed.7, the aorta calcium staining: (1) the Von Kossa staining: the control group rats were stained with each other. At the time point, the aorta was normal. There was no black particle deposition in the aorta of the.CKD group of the black particles, with the rupture of the smooth muscle fibers of the middle membrane and the calcified nodule especially obvious. (2) alizarin red staining: the aorta in the control group was normal at all time points. In the.CKD group, the aorta of the aorta was not seen in the orange red matter group since fourth weeks. The smooth smooth muscle tangerine particles were gradually deposited in the aorta, with the smooth muscle fibers breaking and the calcified nodule deposited more obviously. And with the time progress of the orange red particles, the calcium content of the aorta was gradually increased.8. The calcium content of the aorta was compared with the control group at each time point and the aorta calcium in the CKD group. Content increased significantly (P0.01), and calcium content in group CKD increased gradually (P0.01).9, aorta BMP-2, BMP-4, BMPR-IA (ALK3), MGP immunohistochemical results: the aorta at each time point BMP-2, BMPR-IA, MGP protein in the vascular smooth muscle layer was almost no expression in the control group, and there was a small amount of expression at every time point. In group CKD, the expression of BMP-2, BMPR-IA, MGP in the aorta began to increase obviously on the fourth week of the aorta, and the cytoplasm was brown and yellow, mainly distributed in the smooth muscle cell layer of the middle aorta, and the expression gradually increased with time (P0.01). The expression of BMP-4 protein increased obviously from fourth weeks, sixth weeks and eighth weeks decreased slightly in the fourth week, but with the control group. The same time point was still significantly increased (P0.01).10. Real-time fluorescence quantitative PCR method was used to detect the BMP-2 and BMP-4m RNA expression of the aorta. (1) the expression of BMP-2m RNA in the aorta of the BMP-2m RNA: control group was not significantly changed. The expression of BMP-2m RNA of aorta in group KD increased gradually (P0.01). (2) compared with the control group at each time point, the RNA expression of BMP-4m in CKD group was significantly higher than that in the control group (P0.01) after fourth weeks (P0.01); sixth weeks and 8 weeks were lower than the fourth week (P0.01). Conclusion: (1) adenoopterin combined with high phosphorus diet to feed rats can quickly establish chronic kidney disease. The animal model of vascular calcification (2) BMP-2, BMP-4 and its receptor BMPR-IA (ALK3) in the early stage of vascular calcification in chronic renal disease rats, the protein and gene expression of the inhibitor MGP increased obviously. It indicated that the activation of bone morphogenetic protein related signaling pathway was involved in the development of CKD vascular calcification. (3) the serum BMP-2, BMP-4 water of CKD vascular calcified rats. It has a positive correlation with the degree of aortic calcification, and may be a serum marker for CKD vascular calcification. (4) in the process of vascular calcification in chronic renal disease, the osteogenic activity and expression specificity of BMP-4 may be weaker than that of BMP-2.

【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R692

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