骨形态发生蛋白相关信号通路在慢性肾脏病血管钙化中的作用及机制研究

发布时间：2018-05-06 03:41

本文选题：慢性肾脏病 + 血管钙化　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:慢性肾脏病(Chronic kidney disease,CKD)患者往往合并血管钙化,而血管钙化是其心血管疾病(Cardiovascular disease,CVD)发生率和死亡率增高的主要原因。血管钙化的发生与诸多因素相关,近年来骨形态发生蛋白相关信号通路已成为血管钙化发生机制的研究热点,但其具体机制尚未完全阐明。本研究采用腺嘌呤灌胃联合高磷饲料喂养大鼠,建立CKD血管钙化大鼠模型,观察骨形态发生蛋白相关信号通路,包括骨形态发生蛋白-2(Bone morphogenetic protein-2,BMP-2)、骨形态发生蛋白-4(bone morphogenetic protein-2,BMP-4)、以及受体(Bone morphogenetic protein receptor-IA,BMPR-IA)、抑制剂(Matrix Gla Protein,MGP)在大鼠主动脉上的表达及激活情况,探讨其作用机制,为临床上早期防治CKD血管钙化,降低患者心血管疾病的发病率和死亡率提供策略。方法:(1)CKD血管钙化大鼠模型制备:190-270g左右SPF(无特定病原体)雄性大鼠55只,随机分为对照组(CON,n=20)和CKD组(CKD,n=35),适应性喂养10天后,CKD组给予2.5%腺嘌呤(220-250 mg/kg.d)定时灌胃,第1-4周每日一次,第5-8周隔日一次,联合1.8%高磷大鼠饲料喂养;对照组予以生理盐水(10ml/kg)灌胃,普通大鼠饲料喂养,所有大鼠均自由进食及饮水,造模时间共8周。(2)指标检测:第2、4、6、8周末,分别从CKD组及对照组随机选取6只和5只大鼠处死收集标本。处死前一天,代谢笼收集尿液,检测24小时尿蛋白定量;2%戊巴比妥钠30-60mg/kg腹腔注射麻醉,腹主动脉取血,全自动生化分析仪检测血尿素氮(BUN)、血肌酐(Scr)、胱抑素C(Cysc)、血钙(Ca2+)、血磷(P3-);酶联免疫吸附法(ELISA)测定血清BMP-2、BMP-4含量;测量肾重/体重指数;腹主动脉取血后,快速分离肾脏,置于10%福尔马林液固定,苏木精-伊红(HE)染色法观察肾脏病理改变;快速剥离主动脉,按需分为三部分,一部分用10%中性福尔马林固定,行Von Kossa染色、茜素红染色;免疫组化检测主动脉BMP-2、BMP-4、BMPR-IA(ALK3)、MGP蛋白表达;另一部分,存于装有1ml RNA保护液的EP管中,-20℃冰箱冻存,用于实时荧光定量PCR测定BMP-2、BMP-4 m RNA表达;其余部分于-80℃冰箱保存,用于主动脉钙含量测定。结果:1、一般情况:(1)死亡数:对照组大鼠全部存活;CKD组大鼠第2周出现死亡,死亡1只,实验总过程共死亡9只。(2)大鼠体重、肾重、肾重/体重指数:与对照组各时间点相比,CKD组大鼠体重明显降低(P0.05);而肾重、肾重/体重指数均明显增加(P0.05,P0.05)。2、24h尿蛋白定量:与对照组各时间点相比,CKD组大鼠24h尿蛋白定量明显增加(P0.01),且CKD组大鼠随时间进展24h尿蛋白定量逐渐增加(P0.01)。3、血清学指标:(1)BUN、Scr、Cysc:与对照组相比,CKD组大鼠各时间点血清BUN、Scr、Cysc水平明显增高(P0.01,P0.01,P0.01)。(2)血清钙、磷、钙磷乘积:与对照组相比,CKD组大鼠各时间点血磷及钙磷乘积水平明显增加(P0.01,P0.01),血钙水平自第4周出现明显降低(P0.05)。4、ELISA法测定血清BMP-2、BMP-4含量:(1)BMP-2:与对照组相比,CKD组大鼠各时间点血清BMP-2水平明显增加(P0.05)。此外,CKD组大鼠血清BMP-2随时间进展逐渐增加(P0.05)。(2)BMP-4:与对照组相比较,CKD组大鼠血清BMP-4水平自第4周开始均较同时间点增高(P0.01)。此外,CKD组大鼠血清BMP-4水平第6周、第8周时较第4周降低(P0.01)。5、肾脏病理改变:(1)肉眼:对照组大鼠肾脏大小适中,颜色暗红,表面光滑,质地柔软,皮髓质分界清晰;CKD组大鼠肾脏呈“大白肾”改变,颜色灰白,表面不光滑,弹性差。(2)肾脏HE染色:对照组肾脏形态、结构无异常改变。CKD组自第2周起,出现肾小管扩张,可见小管内棕黄色物质沉积,随时间进展,第4,6,8周逐渐出现肾小球囊腔扩张,间质纤维化,肾小球部分萎缩,炎性细胞浸润,血管减少。6、主动脉形态改变:对照组主动脉光滑,弹性好。CKD组自第4周开始主动脉逐渐弯曲、膨大,呈动脉瘤样改变,弹性降低,第6,8周血管僵硬度明显增加,可见钙化结节形成。7、主动脉钙染色:(1)Von Kossa染色:对照组大鼠各时间点主动脉形态均正常,未见黑色颗粒物沉积。CKD组主动脉自第4周开始出现黑色颗粒沉积,伴中膜平滑肌纤维断裂,钙化结节处尤其明显,且随时间进展黑色颗粒沉积逐渐加重。(2)茜素红染色:对照组大鼠各个时间点主动脉形态均正常,未见橘红色物质沉积。CKD组主动脉自第4周开始逐渐出现中膜平滑肌橘红色颗粒物沉积,伴平滑肌纤维断裂,钙化结节处沉积更明显,且随时间进展橘红色颗粒物沉积逐渐加重。8、主动脉钙含量:与对照组各时间点比较,CKD组大鼠主动脉钙含量明显增加(P0.01),且CKD组内随时间进展钙含量逐渐增加(P0.01)。9、主动脉BMP-2、BMP-4、BMPR-IA(ALK3)、MGP免疫组化结果:对照组大鼠主动脉各时间点BMP-2、BMPR-IA、MGP蛋白在血管中膜平滑肌层几乎无表达,BMP-4在各时间点存在少量表达。与对照组相比,CKD组大鼠第4周起可见主动脉上BMP-2、BMPR-IA、MGP蛋白表达开始明显增加,胞质呈棕黄色,主要分布于主动脉中膜平滑肌细胞层,且随时间进展,其表达逐渐增强(P0.01);BMP-4蛋白自第4周表达明显增加,第6周、第8周较第4周稍下降,但与对照组同时间点比较仍然明显增高(P0.01)。10、实时荧光定量PCR法检测主动脉BMP-2、BMP-4m RNA表达量:(1)BMP-2m RNA:对照组各时间点主动脉BMP-2m RNA的表达无明显变化;与对照组各时间点相比较,CKD组主动脉BMP-2m RNA表达量明显增加(P0.01)。且随时间进展,CKD组主动脉BMP-2m RNA表达逐渐增加(P0.01)。(2)BMP-4m RNA:与对照组各时间点比较,CKD组主动脉BMP-4m RNA表达量自第4周后均明显高于对照组(P0.01);第6周、8周较第4周降低(P0.01)。结论:(1)腺嘌呤灌胃联合高磷饲料喂养大鼠,能够快速建立慢性肾脏病血管钙化动物模型。(2)慢性肾脏病大鼠血管钙化早期即存在BMP-2、BMP-4及其受体BMPR-IA(ALK3)、抑制剂MGP的蛋白及基因表达明显增高,说明骨形态发生蛋白相关信号通路的激活参与CKD血管钙化的发生发展。(3)CKD血管钙化大鼠血清BMP-2、BMP-4水平明显增高,且与主动脉钙化程度呈正相关,可能可作为CKD血管钙化的血清标志物。(4)慢性肾脏病血管钙化过程中,BMP-4的成骨活性及表达特异性可能弱于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.

【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692

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