Fluvastatin抑制肺动脉瓣内皮细胞钙化及其机制研究

发布时间：2018-07-05 13:56

本文选题：血管内皮细胞生长因子 + 肺动脉瓣内皮细胞　；参考：《华中科技大学》2012年博士论文

【摘要】：(第一部分) 背景：血管钙化是矿物质代谢失调在心血管系统的表现。它以病变的动脉壁及瓣膜中大量钙质的沉积和骨样组织形成为标志性病理改变,动脉瓣膜是最容易受累的部位。瓣膜钙化最初被认为是一种老年退行性病变,现已证实是一个主动的炎症、增生和生物矿化的过程。新生微血管和骨样组织形成在瓣膜钙化早期的特征性表现提示了血管内皮细胞生长因子VEGF在瓣膜钙化过程中的重要作用。目的：探讨VEGF是否能够引起瓣膜内皮细胞钙化及其可能的分子机制。方法：取原代培养的人肺动脉瓣内皮细胞,VEGF作用后,以fura-2检测细胞内钙信号；荧光显微镜下观察VEGF刺激后GFP-NFAT融合蛋白的核转位；real-timePCR检测BMP-2、cbfa1、osteocalcin及osteoprotegerin等骨形成相关基因的表达；Von Kossa染色检测细胞钙化。结果：100ng/ml VEGF的刺激激发了人肺动脉瓣内皮细胞中钙振荡的产生,频率为0.3±0.03次/min,波宽80±3.5S,并引起NFAT快速的核转位。BMP-2、cbfa1、 osteocalcin及osteoprotegerin的mRNA水平在刺激后6-24小时均显著提高,并在48小时左右回落。刺激后72小时,在培养单层细胞的基质中发现钙化斑块的出现。结论：VEGF诱导瓣膜内皮细胞骨化相关基因的表达,促进内皮细胞钙化；这种作用与VEGF激发瓣膜内皮细胞钙振荡,进而引发NFAT活化及核转位有关。 (第二部分) 背景：Fluvastatin是一种他汀类药物,是HMG-CoA还原酶的抑制剂,可通过与HMG-CoA还原酶的活性中心结合,阻断甲羟戊酸代谢途径,起到心血管保护作用。目的：探讨fluvastatin是否能够抑制VEGF所造成的瓣膜内皮细胞钙化及其与细胞内钙信号间的关系。方法：培养的人肺动脉内皮细胞以fluvastatin预先处理24小时,再给予100ng/ml VEGF刺激,fura-2检测细胞内钙信号；表达GFP-NFAT融合蛋白的质粒转染细胞,荧光显微镜下观察VEGF刺激后GFP-NFAT的核转位；real-time PCR检测BMP-2、cbfa1、osteocalcin及osteoprotegerin等骨形成相关基因的表达；Von Kossa染色检测细胞钙化。结果：10nM fluvastatin的预处理抑制了瓣膜内皮细胞中钙振荡的产生及后继的NFAT核转位。BMP-2、cbfa1、osteocalcin及osteoprotegerin等骨形成相关基因的mRNA水平分别降低了(50.61±10.88)%,(82.23±22.11)%,(85.58±19.03)%和(85.57±8.42)%。VEGF刺激后72小时,在培养单层细胞的基质中未见钙化斑块。结论：fluvastatin有效抑制了VEGF所引起的瓣膜内皮细胞钙化,且这种作用是通过其抑制细胞内钙信号依赖的NFAT信号通路来实现。 (第三部分) 背景：GGPP是除胆固醇以外被statins阻断的另一类异戊二烯。研究提示statins的多种保护性药理学作用均与对GGPP的合成阻断,从而影响细胞中小G蛋白活性有关。目的：探讨fluvastatin抑制VEGF所致瓣膜内皮细胞钙化的机制方法：培养的人肺动脉内皮细胞以GGPP和fluvastatin共同预处理24小时,再给予100ng/ml VEGF刺激,fura-2检测细胞内钙信号；表达NFAT-GFP融合蛋白的质粒转染细胞,荧光显微镜下观察VEGF刺激后NFAT的核转位；real-time PCR检测BMP-2、cbfal、osteocalcin及osteoprotegerin等骨形成相关基因的表达；Von Kossa染色检测细胞钙化。结果：10nM的GGPP和fluvastatin共同预处理后,VEGF仍然可以诱导瓣膜内皮细胞中钙振荡的产生,频率为0.074±0.02次／min,波宽65.7±8.8S,以及NFAT的核转位。BMP-2、cbfal、osteocalcin及osteoprotegerin的mRNA水平较未补充GGPP前分别提高了1.88±0.16、8.44±3.56、6.59±3.7和2.34±0.16倍。VEGF刺激后72小时,在培养的单层细胞基质中发现钙化斑块的出现。结论：GGPP逆转了fluvastatin对VEGF所致血管内皮细胞钙化的抑制,说明本实验中fluvastatin对血管钙化的保护作用是通过阻断GGPP而实现。
[Abstract]:(Part I)
Background: vascular calcification is a manifestation of the metabolic disorder of the mineral in the cardiovascular system. It is a landmark pathological change in the lesions of the artery wall and a large number of calcium deposits and osteoid tissue in the valve. The artery valve is the most vulnerable part. Valve calcification is originally considered a degenerative disease of the elderly and has been proved to be a main disease. The important role of vascular endothelial growth factor VEGF in valve calcification is suggestive of the early characteristics of the formation of new microvascular and osteoid tissue in the early stage of valve calcification.
Objective: To investigate whether VEGF can induce calcification of valve endothelial cells and its possible molecular mechanism.
Methods: the cultured human pulmonary artery valve endothelial cells were cultured. After VEGF action, the intracellular calcium signal was detected by fura-2; the nuclear translocation of GFP-NFAT fusion protein was observed by VEGF stimulation under the fluorescence microscope; real-timePCR was used to detect the expression of bone morphogenetic genes of BMP-2, Cbfa1, osteocalcin and osteoprotegerin, and Von Kossa staining was used to detect the cells. Calcification.
Results: the stimulation of 100ng/ml VEGF stimulated the production of calcium oscillation in the human pulmonary artery valve endothelial cells, the frequency was 0.3 + 0.03 times /min, the width of the wave was 80 + 3.5S, and the NFAT rapid nuclear transposition.BMP-2, the mRNA levels of Cbfa1, osteocalcin and osteoprotegerin all increased in 6-24 hours after the stimulation, and fell about 48 hours. 72 hours after stimulation. Calcified plaques were found in the matrix of cultured monolayer cells.
Conclusion: VEGF induces the expression of ossification related genes in the valvular endothelial cells and promotes the calcification of endothelial cells, which is related to the activation of NFAT activation and nuclear transposition by VEGF stimulation of the calcium oscillation in the valve endothelial cells.
(the second part)
Background: Fluvastatin is a statin drug, an inhibitor of the HMG-CoA reductase, which can block the metholvalic acid metabolic pathway by combining with the active center of HMG-CoA reductase and play a protective role in cardiovascular protection.
Objective: To investigate whether fluvastatin can inhibit the calcification of valvular endothelial cells caused by VEGF and its relationship with intracellular calcium signaling.
Methods: the cultured human pulmonary artery endothelial cells were pretreated with fluvastatin for 24 hours, then were stimulated by 100ng/ml VEGF, fura-2 was used to detect intracellular calcium signal, the plasmid expressing GFP-NFAT fusion protein was transfected into cells, and the nuclear transposition of GFP-NFAT after VEGF stimulation was observed under the fluorescence microscope; real-time PCR detected BMP-2, Cbfa1, osteocalcin and others. Otegerin and other bone formation related genes were expressed and Von Kossa staining was used to detect cell calcification.
Results: the pretreatment of 10nM fluvastatin inhibited the production of calcium oscillation in the valve endothelial cells and the subsequent NFAT nuclear transposition.BMP-2, and the mRNA level of the bone formation related genes, such as Cbfa1, osteocalcin and osteoprotegerin, decreased (50.61 + 10.88)%, (82.23 + 22.11)%, (85.58 + 19.03)% and (85.57 + 8.42)%.VEGF stimulation for 72 hours. No calcified plaque was found in the matrix of monolayer cells.
Conclusion: fluvastatin effectively inhibits the calcification of valvular endothelial cells caused by VEGF, and this effect is achieved through the NFAT signaling pathway that inhibits the intracellular calcium signal dependence.
(the third part)
Background: GGPP is another class of isoprene that is blocked by statins except cholesterol. The study suggests that various protective pharmacological actions of statins are related to the synthesis of GGPP, which may affect the activity of small and medium G proteins in cells.
Objective: To investigate the mechanism of fluvastatin inhibiting VEGF Induced Calcification of valve endothelial cells.
Methods: the cultured human pulmonary artery endothelial cells were pretreated with GGPP and fluvastatin for 24 hours, then were stimulated by 100ng/ml VEGF, fura-2 was used to detect intracellular calcium signal, the plasmid expressing NFAT-GFP fusion protein was transfected into cells, and the nuclear transfer of NFAT was observed by VEGF stimulation under fluorescence microscope; real-time PCR detection BMP-2, cbfal, etc. Steoprotegerin and other bone formation related genes were expressed and Von Kossa staining was used to detect cell calcification.
Results: after the co pretreatment of 10nM GGPP and fluvastatin, VEGF can still induce the production of calcium oscillation in the valve endothelial cells, the frequency is 0.074 + 0.02 times / min, the width of the wave is 65.7 + 8.8S, and the nuclear transposition of NFAT.BMP-2, cbfal, osteocalcin and osteoprotegerin mRNA levels are increased by 1.88 +. 3.7 and 2.34 + 0.16 times.VEGF, 72 hours after stimulation, calcified plaques were found in cultured monolayer cells.
Conclusion: GGPP reverses the inhibitory effect of fluvastatin on the calcification of vascular endothelial cells induced by VEGF, indicating that the protective effect of fluvastatin on vascular calcification is achieved by blocking the GGPP.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【相似文献】