摘要
目的心肌内血管紧张素系统的激活是心力衰竭发生发展的重要因素之一,而其具体作用机制尚有许多不明之处。近年来心肌微血管新生障碍在心力衰竭发病中的作用逐渐得到认识。本研究观察了血管紧张素Ⅱ对心肌微血管内皮细胞功能的直接影响,为进一步明确血管紧张素Ⅱ引起心力衰竭的机制提供参考。方法(1)植块法分离成年Wistar雄性大鼠的心肌微血管内皮细胞,免疫荧光鉴定细胞种类并传代培养;(2)血管紧张素Ⅱ(10^(-7)-10^(-6)M)干预培养心肌微血管内皮细胞后,分别采用Western blot方法以及PT-PCR的方法,检测不同干预时间点细胞增殖相关蛋白激酶(ERK)的活性变化和细胞死亡相关分子p53的表达变化;(3)血管形成实验检测心肌微血管内皮细胞的功能:血管紧张素Ⅱ干预培养在Matrigel上的微血管内皮细胞后,显微镜下观察形成管腔样结构的能力。结果(1)植块法培养原代心肌微血管内皮细胞,细胞纯度达95%左右,且具有形成管腔样结构的能力;(2)在血管紧张素Ⅱ干预心肌微血管内皮细胞10分钟时,ERK的磷酸化水平明显升高,与非干预组相比具有统计学差异(P<0.05);(3)血管紧张素Ⅱ干预心肌微血管内皮细胞30 min,虽然与非干预组相比没有统计学差异,但p53 mRNA表达增加;(4)用血管紧张素Ⅱ持续干预18 h,心肌微血管内皮细胞形成管腔样结构的数量明显减少,与非干预组相比具有统计学差异(P<0.01);单纯血管紧张素Ⅱ持续干预18小时组与前处置10分钟后再干预18小时组相比,心肌微血管内皮细胞形成管腔样结构的数量明显减少、或基本上不形成管腔样结构,两组相比具有统计学差异(P<0.05)。结论持续血管紧张素Ⅱ干预明显降低培养心肌微血管内皮细胞形成管腔样结构的完整性及其数量,其机制可能与增加p53的表达有关;短期血管紧张素Ⅱ前处置培养心肌微血管内皮细胞明显改善持续血管紧张素Ⅱ干预后细胞形成管腔样结构能力的下降,其机制可能与短期升高细胞内蛋白激酶ERK的活性进而起到细胞保护作用有关。
Objective Local angiotensin system plays an important role in the development of heart failure, but the mechanism of how angiotensin Ⅱ is involved remains unclear. It has been recently reported that dysfunctions of angiogenesis in myocardium are critical to the progress of heart failure. We therefore examined here whether angiotensin Ⅱ might induce dysfunctions of cultured cardiac microvascular endothelial cells and the possible mechanism involved in the effect of angiotensin Ⅱ. Method （1） Myocardial microvascular endothelial cells were cultured by the method of planting myocardial tissue of adult rats and characterized by the immunocytochemistry staining and the capillary tube-like formation; （2）ERK phosphorylation and p53 expression were determined by methods of Western blot and PT-PCR, respectively, after treatment with angiotensin Ⅱ （10^-7-10^- 6 M） in microvascular endothelial cells at different times; （3）The capillary tube-like formation was observed under a microscope after incubation of cardiac microvascular endothelial cells with angiotensin Ⅱ （ 10^-6 M）. Result （1） Purity of microvascular endothelial cells characterized by staining with vWF was about 95%, and the ceils showed a high ability to form the capillary tube-like structures after grown in matrigel for 18 hours ; （2） Phosphorylation of ERKs after addition of angiotensin Ⅱ to the microvascular endothelial cells for 10 min was significantly increased than those without angiotensin Ⅱ addition （ P 〈 0.05 ） ; （ 3 ） The gene expression of p53 was slightly enhanced by stimulation with angiotensin Ⅱ for 30 min as compared to those without the stimulation. 4. The formation of capillary-like tubes by microvascular endothelial cells was decreased after incubation with angiotensin Ⅱ for 18 hours as compared to those without angitensin Ⅱ treatment （P 〈0. 05 or P 〈 0. 01 ） ; However, it was significantly improved when the cells were pretreated with angiotensin for 10 min before a long time （ 18 hours） incubation comparing to those with angiotensin Ⅱ treatment for 18 hour alone. Conclusion Persistent treatment with angiotensin Ⅱ for a long time significantly impaired the ability of cardiac microvascular endothelial cells to form the capillary tube-like structures, which might associate with the upregulation of p53 expression; Pretreatment for a short time before the long term treatment with angiotensin Ⅱ significantly improved the dysfunctions of microvascular endothelial ceils induced by the treatment with angiotensin Ⅱ , which might be attributed to a transient increase in the activities of ERKs and the protection of the cells by ERKs.
出处
《中国分子心脏病学杂志》
CAS
2009年第1期1-6,共6页
Molecular Cardiology of China
基金
国家"973"项目子课题:2007CB512003
高等学校博士学科点专项科研基金:20060246079
