内皮素-1调控主动脉瓣膜间质细胞向成骨细胞分化作用机制研究

发布时间：2018-03-04 15:36

本文选题：主动脉瓣膜间质细胞　切入点：成骨细胞分化　出处：《中南大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：分离并培养出人主动脉瓣膜间质细胞并利用钙化培养基进行干预建立其向成骨细胞分化的细胞模型方法：收集sanford A型主动脉夹层患者(夹层撕裂累及主动脉瓣)的主动脉瓣叶采用胶原酶消化法,分离出瓣膜间质细胞。免疫组化鉴定细胞表型：平滑肌α肌动蛋白,波形蛋白,CD31,平滑肌肌球蛋白和结蛋白。同时对细胞使用钙化培养基(含β甘油磷酸钠,地塞米松和维生素C)促进主动脉瓣膜间质细胞向成骨细胞分化。通过碱性磷酸酶活性检测、染色和钙化结节染色(von Kossa法)以及western blot检测碱性磷酸酶(ALP)及核结合因子α-1(Cbf a1)的表达评估该模型的效果结果：分离出的主动脉瓣膜间质细胞能够增殖,并表达平滑肌α肌动蛋白和波形蛋白,但不表达CD31,平滑肌肌球蛋白和结蛋白。在钙化培养基干预后的第21天,模型组较对照组的碱性磷酸酶活性、染色和钙化结节染色更明显,且表达更多的ALP和Cbfal。结论：胶原酶消化法分离的人主动脉瓣膜间质细胞能够在体外条件下增殖,并表达特有的表型,同时钙化培养基能够明显地促进主动脉瓣膜间质细胞向成骨细胞分化,ALP和Cbfal的表达并形成钙化结节。目的：证实内皮素-1能够促进主动脉瓣膜间质细胞向成骨细胞分化,并讨论内皮素受体A (ETAR)和细胞外信号调节蛋白激酶(ERK)通路在其中扮演的角色。方法：在钙化培养基中分别添加不同浓度的内皮素-1或内皮素-1+ERK通路阻滞剂(PD98059)或内皮素-1+ETAR阻滞剂(BQ-123)。在干预的第21天分别检测碱性磷酸酶的活性和表达水平。干预的第21天检测钙化结节,并检测表型蛋白的变化。在培养基中添加内皮素-1后,在不同时间(5min,10min,30min,60min,120min)检测ERK通路的激活情况。在培养基中添加内皮素-1和ETAR阻滞剂BQ-123和ERK通路阻滞剂PD98059,干预20分钟后检测ERK通路的激活程度。结果：内皮素-1以浓度依赖性促进主动脉瓣膜间质细胞产生碱性磷酸酶,BQ-123及PD98059能够明显抑制内皮素-1的上述作用。内皮素-1可明显促进钙化结节的产生及激活ERK通路,BQ-123和PD98059能够抑制内皮素-1对该通路的激活。结论：内皮素-1通过ETAR受体和ERK通路以浓度依赖性促进主动脉瓣膜间质细胞向成骨细胞分化。
[Abstract]:Objective: to isolate and culture human aortic valve mesenchymal cells and to establish a cell model of osteoblast differentiation using calcification medium. Methods: the interstitial cells of aortic valve were isolated from aortic valve of sanford A aortic dissection by collagenase digestion. The phenotype of the cells was identified by immunohistochemistry: smooth muscle 伪 actin. Vimentin CD31, smooth muscle myosin and desmin. At the same time, calcification medium (containing 尾 -glycerophosphate, 尾 -glycerophosphate, 尾 -glycerophosphate) was used for cells. Dexamethasone and vitamin C) promote the differentiation of aortic valve interstitial cells into osteoblasts. The expression of alkaline phosphatase (ALP) and nuclear binding factor 伪 -1 (Cbfa1) were evaluated by staining and calcified nodule staining (von Kossa) and western blot to evaluate the effect of the model. Results: the isolated aortic valve stromal cells could proliferate and express smooth muscle 伪 actin and vimentin, but not CD31, smooth muscle myosin and desmin. The activity of alkaline phosphatase, staining and calcified nodule staining in model group were more obvious than those in control group, and the expression of ALP and Cbfalin were higher in the model group than in the control group. Conclusion: human aortic valve mesenchymal cells isolated by collagenase digestion can proliferate and express specific phenotypes in vitro. At the same time, calcification medium could obviously promote the expression of ALP and Cbfal in the differentiation of aortic valve interstitial cells into osteoblasts and form calcified nodules. Aim: to demonstrate that endothelin-1 can promote the differentiation of aortic valve interstitial cells into osteoblasts, and to discuss the roles of endothelin receptor A (ETAR) and extracellular signal regulated protein kinase (ERK) pathway in the differentiation of aortic valve interstitial cells into osteoblasts. Methods: different concentrations of endothelin-1 or endothelin-1 ERK pathway blockers (PD98059) or endothelin-1 ETAR blockers (BQ-123) were added to calcified media. The activity and expression of alkaline phosphatase were detected on the 21st day of intervention. ... on the 21st day of intervention, calcified nodules were detected, The changes of phenotypic protein were detected. The activation of ERK pathway was detected at different time points (5min / 10min / 30min / 60min / 120min). The activation of ERK pathway was detected by adding endothelin-1 and ETAR blocker BQ-123 and ERK pathway blocker PD98059. after 20 minutes of intervention, the activation of ERK pathway was detected. Results: endothelin-1 enhanced alkaline phosphatase production by aortic valve interstitial cells in a concentration-dependent manner. BQ-123 and PD98059 could significantly inhibit the above effects of endothelin-1. Endothelin-1 significantly promoted the production and activation of calcified nodules. ERK pathway BQ-123 and PD98059 can inhibit the activation of endothelin-1. Conclusion: endothelin-1 promotes the differentiation of aortic valve interstitial cells into osteoblasts in a concentration-dependent manner through ETAR receptor and ERK pathway.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

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