模拟失重对肺微血管内皮细胞的影响
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摘要
失重或模拟失重可导致肺循环功能失调,血管结构、功能的变化在其中具有重要作用。肺微血管内皮细胞功能对于肺循环的正常功能维持具有重要作用,是构成微血管通透性的主要物理屏障,不仅能保证微血管内外正常的物质交换,而且可分泌多种因子调节微血管的舒缩功能。失重或模拟失重后肺微血管内皮细胞结构、功能变化及其内在机制并不清楚。因此,本课题的目的为：
(1)观察回转模拟失重对肺微血管内皮细胞超微结构的影响；
(2)研究回转模拟失重对肺微血管内皮细胞合成NO产量及eNOS蛋白表达的影响；
(3)研究回转模拟失重对肺微血管内皮细胞骨架的影响；
(4)研究回转模拟失重对肺微血管内皮细胞凋亡的影响初步分子机制。
方法
采用组织块贴壁法培养原代肺微血管内皮细胞,以倒置相差显微镜、Ⅷ因子免疫荧光染色、扫描电镜和透射电镜等方法对培养的肺微血管内皮细胞进行鉴定；采用回转器回转细胞模拟失重,以扫描电镜和透射电镜法对回转模拟失重后肺微血管内皮细胞的超微结构变化进行观察;以Griess法对培养的肺微血管内皮细胞合成释放的NO浓度进行检测,以免疫组化技术和Western blot技术对肺微血管内皮细胞中的eNOS蛋白表达进行检测；以Annexin V-FITC和PI双染标记细胞,采用流式细胞术及Hoechst33258细胞核染色对肺微血管内皮细胞凋亡进行检测：采用免疫荧光标记技术,以Texas Red-X偶联的phalloidin标记肺微血管内皮细胞微丝肌动蛋白,观察回转模拟失重对肺微血管内皮细胞骨架的影响；采用放免技术检测回转模拟失重后肺微血管内皮细胞ET-1合成释放量的变化,以Western blot技术和免疫荧光技术检测肺微血管内皮细胞Akt磷酸化水平的变化及P21Cip1蛋白表达的变化；采用Western blot技术对凋亡调控蛋白Bcl-2和Bax的表达进行检测。
结果
扫描电镜结果表明,回转48h后,肺微血管内皮细胞单层细胞之间的连接较为松散,可见有明显的裂缝存在；而在同步对照组的肺微血管内皮细胞单层可见细胞之间的连接较为紧密,未见裂缝或空洞存在,说明回转模拟失重能够破坏肺微血管内皮细胞单层,导致肺微血管内皮细胞单层的通透性明显增强。透射电镜结果表明,回转48小时后,可见肺微血管内皮细胞中线粒体增生、肿胀。NO检测及Western blot、免疫组化结果表明,回转模拟失重可导致肺微血管内皮细胞的NO产量增加,同时增强eNOS蛋白的表达,并且这种增加存在时间依赖关系。Hoechst33258染色及流式细胞术检测结果表明,回转48h模拟失重肺微血管内皮细胞凋亡率明显高于对照；phalloidin肺微血管内皮细胞微丝肌动蛋白染色结果表明,回转模拟失重可导致微丝细胞骨架随着回转时间的延长出现进行性的解聚：回转48h后,放免结果表明肺微血管内皮细胞合成释放ET-1的量下降；Western blot及免疫荧光染色结果表明,Akt磷酸化水平下降,而P21Cip1蛋白表达增强;Western blot检测凋亡相关蛋白结果表明,回转模拟失重后肺微血管内皮细胞中抗凋亡蛋白Bcl-2表达显著下降,而促凋亡蛋白Bax表达显著升高。
结论
(1)回转模拟失重可导致肺微血管内皮细胞中线粒体增生、肿胀;破坏肺微血管内皮细胞单层,导致肺微血管内皮细胞单层的通透性明显增强；
(2)回转模拟失重可刺激肺微血管内皮细胞中NO的合成和释放,增强eNOS蛋白的表达,并且这种eNOS表达的增强和NO产量的增加与回转模拟失重的时间长短呈正相关；
(3)回转模拟失重可导致肺微血管内皮细胞发生早期凋亡；
(4)回转模拟失重可导致PMVEC内微丝细胞骨架发生解聚;
(5)回转模拟失重导致了肺微血管内皮细胞ET-1合成分泌下降及线粒体膜通透性转换孔开放增加；抑制肺微血管内皮细胞中Akt的磷酸化水平,增强P21Cip1蛋白的核内表达水平；抑制凋亡抑制蛋白Bcl-2的表达,增强了促凋亡蛋白Bax的表达。
Objective
Microgravity or simulated microgravity can induce dysfunction of pulmonary circulation, in which the changes of vascular structure and function have important role. The function of pulmonary microvascular endothelial cells play important role in maintaining the normal functions of pulmonary circulation. Moreover, pulmonary microvascular endothelial cells compose the main physical barrier for microvascular permeability, which can not only guarantee the normal substance exchange in microvascular, but also excrete all kinds of factors regulating the contractile and relaxing function of microvascular. However, there is not any report concerning the effects of microgravity or simulated microgravity on the function of pulmonary microvascular endothelial cells until now, the mechanisms of changes for pulmonary microvascular endothelial cells structure and function are not clear. So, the purpose of this study is following::
(1) Observe effects of simulated microgravity induced by clinostat on ultrastructure of pulmonary microvascular endothelial cells.
(2) Study the effects of simulated microgravity induced by clinostat on NO production and protein expression of eNOS in pulmonary microvascular endothelial cells.
(3) Study the effects of simulated microgravity on cytoskeleton of pulmonary microvascular endothelial cells.
(4) Study the effects of simulated microgravity on apoptosis in pulmonary microvasuclar endothelial cells and its molecular mechanisms.
Methods
Pulmonary microvascular endothelial cells were cultured by implanting tissue pieces and were identified by cell morphology under inverted phase contrast microscope,Ⅷfactor immunofluorescence staining, scanning electron microscopy and transmission electron microscopy. Simulated microgravity was induced by clinostat, scanning and transmission electron microscopy were used to observe the ultrastructure of pulmonary microvascular endothelial cells. NO concentration released by pulmonary microvascular endothelial cells was measured by Griess method, eNOS protein expression was measured by western blot and immunostaining methods. AnnexinⅤ-FITC and PI were used to label cells, apoptosis of pulmonary microvascular endothelial cells was detected by nucleus staining with Hoechst 33258 and flow cytometry. By immunofluorescence technique, phalloidin labeled by Texas Red-X was used to incubate with pulmonary microvascular endothelial cells to detect the microfilament cytoskeleton. Radioimmunoassay technique was used to detect the ET-1 concentration released by pulmonary microvascular endothelial cells, western blot and immunofluorescence methods were used to measure the changes of Akt phosphorylation and P21Cip1 protein expression. Protein expression of apoptosis regulating factors Bcl-2 and Bax were measured by western blot.
Results
The results from sanning electron microscopy showed that the contacts of pulmonary microvascular endothelial cells became incompact and obvious crack appeared after 48h clinostat, which were not observed in control pulmonary microvascular endothelial cells. These results illuminated that simulated microgravity can destroy the cell monolayer of pulmonary microvascular endothelial cells and induced the increased permeability of cell monolayer. The results from transmission electron microscopy showed that the mitochondrion became hyperplasia and swelling. The results of NO measurement, western blot and immunostaining showed that simulated microgravity induced the increased NO production and eNOS protein expression which were dependent with time of clinostat. The results from Hoechst33258 and flow cytometry showed that 48h clinostat induced the increased apoptosis rate of pulmonary microvascular endothelial cells. Microfilament staining by phalloidin showed that simulated microgravity induced microfilament depolymerization which was progressive with time of clinostat. After 48h clinostat, the results from radioimmunoassay showed that ET-1 released by pulmonary microvascular endothelial cells was lower than that of control. The results of western blot and immunofluorescence showed that the phosphorylation level of Akt was decreased, the protein expression of P21Cip1 was increased. The measurements of apoptosis related proteins by western blot showed that simulated microgravity significantly decreased the protein expression of Bcl-2 but increased the expression of Bax protein.
Conclusion
(1) Simulated microgravity induces mitochondrion hyperplasia and swelling in pulmonary microvascular endothelial cells, destroys the cell monolayer of pulmonary microvascular endothelial cells and induced the increased permeability of cell monolayer.
(2) Simulated microgravity stimulates NO synthesis and releaseing, increases the protein expression of eNOS, which are dependent with time of clinostat.
(3) Simulated microgravity induces early apoptosis of pulmonary microvascular endothelial cells.
(4) Simulated microgravity induces microfilament cytoskeleton depolymerization in pulmonary microvascular endothelial cells.
(5) Simulated microgravity decreases the secretion of ET-1, increases the opening of mPTP, inhibits the phosphorylation level of Akt, increases the protein expression of P21Cip1 in pulmonary microvascular endothelial cells. Moreover, the protein expression of Bcl-2 was decreased and Bax was increased by simulated microgravity induced 48h clinostat.
(1)观察回转模拟失重对肺微血管内皮细胞超微结构的影响；
(2)研究回转模拟失重对肺微血管内皮细胞合成NO产量及eNOS蛋白表达的影响；
(3)研究回转模拟失重对肺微血管内皮细胞骨架的影响；
(4)研究回转模拟失重对肺微血管内皮细胞凋亡的影响初步分子机制。
方法
采用组织块贴壁法培养原代肺微血管内皮细胞,以倒置相差显微镜、Ⅷ因子免疫荧光染色、扫描电镜和透射电镜等方法对培养的肺微血管内皮细胞进行鉴定；采用回转器回转细胞模拟失重,以扫描电镜和透射电镜法对回转模拟失重后肺微血管内皮细胞的超微结构变化进行观察;以Griess法对培养的肺微血管内皮细胞合成释放的NO浓度进行检测,以免疫组化技术和Western blot技术对肺微血管内皮细胞中的eNOS蛋白表达进行检测；以Annexin V-FITC和PI双染标记细胞,采用流式细胞术及Hoechst33258细胞核染色对肺微血管内皮细胞凋亡进行检测：采用免疫荧光标记技术,以Texas Red-X偶联的phalloidin标记肺微血管内皮细胞微丝肌动蛋白,观察回转模拟失重对肺微血管内皮细胞骨架的影响；采用放免技术检测回转模拟失重后肺微血管内皮细胞ET-1合成释放量的变化,以Western blot技术和免疫荧光技术检测肺微血管内皮细胞Akt磷酸化水平的变化及P21Cip1蛋白表达的变化；采用Western blot技术对凋亡调控蛋白Bcl-2和Bax的表达进行检测。
结果
扫描电镜结果表明,回转48h后,肺微血管内皮细胞单层细胞之间的连接较为松散,可见有明显的裂缝存在；而在同步对照组的肺微血管内皮细胞单层可见细胞之间的连接较为紧密,未见裂缝或空洞存在,说明回转模拟失重能够破坏肺微血管内皮细胞单层,导致肺微血管内皮细胞单层的通透性明显增强。透射电镜结果表明,回转48小时后,可见肺微血管内皮细胞中线粒体增生、肿胀。NO检测及Western blot、免疫组化结果表明,回转模拟失重可导致肺微血管内皮细胞的NO产量增加,同时增强eNOS蛋白的表达,并且这种增加存在时间依赖关系。Hoechst33258染色及流式细胞术检测结果表明,回转48h模拟失重肺微血管内皮细胞凋亡率明显高于对照；phalloidin肺微血管内皮细胞微丝肌动蛋白染色结果表明,回转模拟失重可导致微丝细胞骨架随着回转时间的延长出现进行性的解聚：回转48h后,放免结果表明肺微血管内皮细胞合成释放ET-1的量下降；Western blot及免疫荧光染色结果表明,Akt磷酸化水平下降,而P21Cip1蛋白表达增强;Western blot检测凋亡相关蛋白结果表明,回转模拟失重后肺微血管内皮细胞中抗凋亡蛋白Bcl-2表达显著下降,而促凋亡蛋白Bax表达显著升高。
结论
(1)回转模拟失重可导致肺微血管内皮细胞中线粒体增生、肿胀;破坏肺微血管内皮细胞单层,导致肺微血管内皮细胞单层的通透性明显增强；
(2)回转模拟失重可刺激肺微血管内皮细胞中NO的合成和释放,增强eNOS蛋白的表达,并且这种eNOS表达的增强和NO产量的增加与回转模拟失重的时间长短呈正相关；
(3)回转模拟失重可导致肺微血管内皮细胞发生早期凋亡；
(4)回转模拟失重可导致PMVEC内微丝细胞骨架发生解聚;
(5)回转模拟失重导致了肺微血管内皮细胞ET-1合成分泌下降及线粒体膜通透性转换孔开放增加；抑制肺微血管内皮细胞中Akt的磷酸化水平,增强P21Cip1蛋白的核内表达水平；抑制凋亡抑制蛋白Bcl-2的表达,增强了促凋亡蛋白Bax的表达。
Objective
Microgravity or simulated microgravity can induce dysfunction of pulmonary circulation, in which the changes of vascular structure and function have important role. The function of pulmonary microvascular endothelial cells play important role in maintaining the normal functions of pulmonary circulation. Moreover, pulmonary microvascular endothelial cells compose the main physical barrier for microvascular permeability, which can not only guarantee the normal substance exchange in microvascular, but also excrete all kinds of factors regulating the contractile and relaxing function of microvascular. However, there is not any report concerning the effects of microgravity or simulated microgravity on the function of pulmonary microvascular endothelial cells until now, the mechanisms of changes for pulmonary microvascular endothelial cells structure and function are not clear. So, the purpose of this study is following::
(1) Observe effects of simulated microgravity induced by clinostat on ultrastructure of pulmonary microvascular endothelial cells.
(2) Study the effects of simulated microgravity induced by clinostat on NO production and protein expression of eNOS in pulmonary microvascular endothelial cells.
(3) Study the effects of simulated microgravity on cytoskeleton of pulmonary microvascular endothelial cells.
(4) Study the effects of simulated microgravity on apoptosis in pulmonary microvasuclar endothelial cells and its molecular mechanisms.
Methods
Pulmonary microvascular endothelial cells were cultured by implanting tissue pieces and were identified by cell morphology under inverted phase contrast microscope,Ⅷfactor immunofluorescence staining, scanning electron microscopy and transmission electron microscopy. Simulated microgravity was induced by clinostat, scanning and transmission electron microscopy were used to observe the ultrastructure of pulmonary microvascular endothelial cells. NO concentration released by pulmonary microvascular endothelial cells was measured by Griess method, eNOS protein expression was measured by western blot and immunostaining methods. AnnexinⅤ-FITC and PI were used to label cells, apoptosis of pulmonary microvascular endothelial cells was detected by nucleus staining with Hoechst 33258 and flow cytometry. By immunofluorescence technique, phalloidin labeled by Texas Red-X was used to incubate with pulmonary microvascular endothelial cells to detect the microfilament cytoskeleton. Radioimmunoassay technique was used to detect the ET-1 concentration released by pulmonary microvascular endothelial cells, western blot and immunofluorescence methods were used to measure the changes of Akt phosphorylation and P21Cip1 protein expression. Protein expression of apoptosis regulating factors Bcl-2 and Bax were measured by western blot.
Results
The results from sanning electron microscopy showed that the contacts of pulmonary microvascular endothelial cells became incompact and obvious crack appeared after 48h clinostat, which were not observed in control pulmonary microvascular endothelial cells. These results illuminated that simulated microgravity can destroy the cell monolayer of pulmonary microvascular endothelial cells and induced the increased permeability of cell monolayer. The results from transmission electron microscopy showed that the mitochondrion became hyperplasia and swelling. The results of NO measurement, western blot and immunostaining showed that simulated microgravity induced the increased NO production and eNOS protein expression which were dependent with time of clinostat. The results from Hoechst33258 and flow cytometry showed that 48h clinostat induced the increased apoptosis rate of pulmonary microvascular endothelial cells. Microfilament staining by phalloidin showed that simulated microgravity induced microfilament depolymerization which was progressive with time of clinostat. After 48h clinostat, the results from radioimmunoassay showed that ET-1 released by pulmonary microvascular endothelial cells was lower than that of control. The results of western blot and immunofluorescence showed that the phosphorylation level of Akt was decreased, the protein expression of P21Cip1 was increased. The measurements of apoptosis related proteins by western blot showed that simulated microgravity significantly decreased the protein expression of Bcl-2 but increased the expression of Bax protein.
Conclusion
(1) Simulated microgravity induces mitochondrion hyperplasia and swelling in pulmonary microvascular endothelial cells, destroys the cell monolayer of pulmonary microvascular endothelial cells and induced the increased permeability of cell monolayer.
(2) Simulated microgravity stimulates NO synthesis and releaseing, increases the protein expression of eNOS, which are dependent with time of clinostat.
(3) Simulated microgravity induces early apoptosis of pulmonary microvascular endothelial cells.
(4) Simulated microgravity induces microfilament cytoskeleton depolymerization in pulmonary microvascular endothelial cells.
(5) Simulated microgravity decreases the secretion of ET-1, increases the opening of mPTP, inhibits the phosphorylation level of Akt, increases the protein expression of P21Cip1 in pulmonary microvascular endothelial cells. Moreover, the protein expression of Bcl-2 was decreased and Bax was increased by simulated microgravity induced 48h clinostat.
引文
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4. Vaziri, N. D., Y. Ding, D. S. Sangha, et al. Upregulation of NOS by simulated microgravity, potential cause of orthostatic intolerance [J]. J Appl Physiol,2000, 89:338-344
5. Ma Jin, Chadi I. Kahwaji, Zhenmin Ni, et al. Effects of simulated microgravity on arterial nitric oxide synthase and nitrate and nitrite content[J]. J Appl Physiol,2003, 94:83-92
6. ZHANG Hua, SUN Ye qing, ZHANG Feng yun, et al. Effects of simulated microgravity on morphology of human umbilical Vein endothelial cells and its production of NO[J]. Space Medicine &Medical Engineering,2005,18 (5):324-328
7. Masataka Mori. Regulation of Nitric Oxide Synthesis and Apoptosis by Arginase and Arginine Recycling[J]. J. Nutr.2007,137:1616S-1620S
8.Infanger.M, Kossmehl.P, Shakibaei.M, et al. Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity:Impact of vascular endothelial growth factor[J]. Apoptosis,2006,11: 749-764
1. Prisk, GK. Invited Review:Microgravity and the lung[J]. J Appl Physiol,2000,89: 385-396
2. Montmerle, Ste'phanie, Jonas Spaak, and Dag Linnarsson. Lung function during and after prolonged headdown bed rest[J]. J Appl Physiol,2002,92:75-83
3. Baranov VM, Tikhonov MA, and Kotov AN. The external respiration and gas exchanges in space missions[J]. Acta Astronaut,1992,27:45-50
4. Vermes I, Haanen C, Nakken H, et al. A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V[J]. Jour Immu Meth,1995,184:39-51
5. Infanger.M, Kossmehl.P, Shakibaei.M. Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity:Impact of vascular endothelial growth factor[J]. Apoptosis,2006,11(5):749-764
6. Infanger.M, Ulbrich.C, Baatout.S. Modeled gravitational unloading induced downregulation of endothelin-1 in human endothelial cells[J]. J Cell Biochem, 2007,101(6):1439-1455
7. Ulbrich.C, Westphal.K, Baatout.S. Effects of basic fibroblast growth factor on endothelial cells under conditional of simulated microgravity[J]. J Cell Biochem, 2008,104(4):1324-1341
8.岳勇,姚永杰,王春梅,等.模拟失重对家兔股静脉壁超微结构的影响[J].中华航空航天医学杂志,2002,13(4)：226-229
9.郝从军,刘长庭,王俊锋.模拟失重对大鼠肺动脉eNOS表达的影响[J].解放军医学杂志,2006,31(7)：704-705
10.王林林,魏文宁,陈智超.一氧化氮对血管内皮细胞凋亡的影响[J].中国血栓与止血学杂志,2002,8(4)：162-164
1. Ingber, DE. How cells(might) sense microgravity. FASEB J,1999,13(Suppl):S3-15
2. Papaseit C, Pochon N, Tabony J. Microtubule self-organization is Gravity-dependent[J]. Proc Natl Acad Sci USA,2000,97(15):8364-8368
3.王俊锋刘长庭刘岩等.模拟失重对大鼠肺微血管通透性的影响[J].军医进修学院学报,2003,24(1)：13-15
4. Ingber DE. How cells(might) sense microgravity [J]. FASEB J,1999,13 (Suppl):S3-15
5. Versari S, Villa A, Bradamante S,et al. Alterations of the actin cytoskeleton and increased nitric oxide synthesis are common features in human primary endothelial cell response to changes in gravity[J]. Biochim Biophys Acta,2007,1773(11): 1645-52
6. Carlsson SI, Bertilaccio.MT, Ballabio E, et al. Endothelial stress by gravitational unloading:effects on cell growth and cytoskeletal organization[J]. Biochim Biophys Acta,2003,1642(3):173-179
7. Buravkova L, Romanor Y, Rykova.M, et al. Cell to cell interactions in changed gravity:ground-based and flight experiments [J]. Acta Astronaut,2005,57(2-8):67-74
8. Gruener R, Roberts R, Roistetter R. Reduced receptor aggregation and altered cytoskeleton in cultured myocytes after spaceflight[J]. Biol Sci Space, 1994,8(2):79-93
9. Schatten H, Lewis ML, Chakrabarti A. Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells [J]. Acta Astronaut,2001,49(3-10):399-418
1. Infanger.M, Kossmehl.P, Shakibaei.M. Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity[J]:Impact of vascular endothelial growth factor. Apoptosis, 2006,11(5):749-764
2. Infanger.M, Ulbrich.C, Baatout.S. Modeled gravitational unloading induced downregulation of endothelin-1 in human endothelial cells[J]. J Cell Biochem, 2007,101(6):1439-1455
3. Ulbrich.C, Westphal.K, Baatout.S. Effects of basic fibroblast growth factor on endothelial cells under conditional of simulated microgravity[J]. J Cell Biochem, 2008,104(4):1324-1341
4.林跃辉,王敏.PI3K-AKT信号转导途径与凋亡的关系[J] 国际病理科学与临 床杂志,2005,25(4)：307-310
5.王静,李东野,夏勇 转染Akt1基因对缺血再灌注大鼠心肌线粒体通透性转换的影响 中国病理生理杂志,2010,26(1)：80-85
6. Jianhua Feng, Eliana Lucchinetti, Preeti Ahuja, et al. Isoflurane postconditioning prevents opening of the mitochondrial permeability transition pore through inhibition of glycogen synthase kinase 3β. Anesthesiology,2005,103:987-995
7.宋其蔓,孙洞箫细胞凋亡与P21关系的研究 医学综述,2008,14(6)：816-818
8.韩凯,马彦卓,许旭东.内皮素-1对大鼠骨髓内皮前体细胞凋亡和细胞周期的影响[J].第四军医大学学报,2008,29(22)：2026-2028
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12. Hirokazu Ohashi, Hitoshi Takagi, Hideyasu Oh, et al. Phosphatidylinositol 3 Kinase/Akt regulates Angiotensin Ⅱ-induced inhibition of apoptosis in microvascular endothelial cells by governing surviving expression and suppression of caspase-3 activity[J]. Circ Res,2004,94:785-793
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1. Chen SF,Fei X,Li SH. A new simple method for Isolation of microvascular edndothelial cells avoiding both chemical and mechanical injuries[J]. Microvasc Res,1995,50(1):119-128
2.姜明,金岩,刘源.大鼠肺微血管内皮细胞培养方法的研究[J].实用口腔医学杂志,2004,20(1)：24-26
3.李发琪,杨瑞芳,秦健.大鼠肺微血管内皮细胞培养及其粘弹性研究[J].生物医学工程学杂志,2002,19(1)：36-39
4.康辉,高魏,陈元.肺微血管内皮细胞培养的改进方法[J].南京军医学院学报,2002,24(2)：112-113
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7.徐顺贵,吴国明,徐智.组织块法培养大鼠肺微血管内皮细胞的综合鉴定[J].第三军医大学学报,2007,29(1)：39-42
8.王俊锋,刘长庭,刘岩,等.模拟失重对大鼠肺微血管通透性的影响[J].军医进修学院学报,2003,24(1)：13-15
9.岳勇,姚永杰,王春梅,等.模拟失重对家兔股静脉壁超微结构的影响[J].中华航空航天医学杂志,2002,13(4)：226-229
1. Prisk GK. Physiology of a Microgravity Environment Invited Review:Microgravity and the lung[J]. JAppl Physiol,2000,89:385-396
2.Montmerle, Ste'phanie, Jonas Spaak, and Dag Linnarsson. Lung function during and after prolonged headdown bed rest[J]. J Appl Physiol,2002,92:75-83
3 Baranov VM, Tikhonov MA, and Kotov AN. The external respiration and gas exchanges in space missions[J]. Acta Astronaut,1992,27:45-50
4. Vaziri, N. D., Y. Ding, D. S. Sangha, et al. Upregulation of NOS by simulated microgravity, potential cause of orthostatic intolerance [J]. J Appl Physiol,2000, 89:338-344
5. Ma Jin, Chadi I. Kahwaji, Zhenmin Ni, et al. Effects of simulated microgravity on arterial nitric oxide synthase and nitrate and nitrite content[J]. J Appl Physiol,2003, 94:83-92
6. ZHANG Hua, SUN Ye qing, ZHANG Feng yun, et al. Effects of simulated microgravity on morphology of human umbilical Vein endothelial cells and its production of NO[J]. Space Medicine &Medical Engineering,2005,18 (5):324-328
7. Masataka Mori. Regulation of Nitric Oxide Synthesis and Apoptosis by Arginase and Arginine Recycling[J]. J. Nutr.2007,137:1616S-1620S
8.Infanger.M, Kossmehl.P, Shakibaei.M, et al. Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity:Impact of vascular endothelial growth factor[J]. Apoptosis,2006,11: 749-764
1. Prisk, GK. Invited Review:Microgravity and the lung[J]. J Appl Physiol,2000,89: 385-396
2. Montmerle, Ste'phanie, Jonas Spaak, and Dag Linnarsson. Lung function during and after prolonged headdown bed rest[J]. J Appl Physiol,2002,92:75-83
3. Baranov VM, Tikhonov MA, and Kotov AN. The external respiration and gas exchanges in space missions[J]. Acta Astronaut,1992,27:45-50
4. Vermes I, Haanen C, Nakken H, et al. A novel assay for apoptosis flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V[J]. Jour Immu Meth,1995,184:39-51
5. Infanger.M, Kossmehl.P, Shakibaei.M. Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity:Impact of vascular endothelial growth factor[J]. Apoptosis,2006,11(5):749-764
6. Infanger.M, Ulbrich.C, Baatout.S. Modeled gravitational unloading induced downregulation of endothelin-1 in human endothelial cells[J]. J Cell Biochem, 2007,101(6):1439-1455
7. Ulbrich.C, Westphal.K, Baatout.S. Effects of basic fibroblast growth factor on endothelial cells under conditional of simulated microgravity[J]. J Cell Biochem, 2008,104(4):1324-1341
8.岳勇,姚永杰,王春梅,等.模拟失重对家兔股静脉壁超微结构的影响[J].中华航空航天医学杂志,2002,13(4)：226-229
9.郝从军,刘长庭,王俊锋.模拟失重对大鼠肺动脉eNOS表达的影响[J].解放军医学杂志,2006,31(7)：704-705
10.王林林,魏文宁,陈智超.一氧化氮对血管内皮细胞凋亡的影响[J].中国血栓与止血学杂志,2002,8(4)：162-164
1. Ingber, DE. How cells(might) sense microgravity. FASEB J,1999,13(Suppl):S3-15
2. Papaseit C, Pochon N, Tabony J. Microtubule self-organization is Gravity-dependent[J]. Proc Natl Acad Sci USA,2000,97(15):8364-8368
3.王俊锋刘长庭刘岩等.模拟失重对大鼠肺微血管通透性的影响[J].军医进修学院学报,2003,24(1)：13-15
4. Ingber DE. How cells(might) sense microgravity [J]. FASEB J,1999,13 (Suppl):S3-15
5. Versari S, Villa A, Bradamante S,et al. Alterations of the actin cytoskeleton and increased nitric oxide synthesis are common features in human primary endothelial cell response to changes in gravity[J]. Biochim Biophys Acta,2007,1773(11): 1645-52
6. Carlsson SI, Bertilaccio.MT, Ballabio E, et al. Endothelial stress by gravitational unloading:effects on cell growth and cytoskeletal organization[J]. Biochim Biophys Acta,2003,1642(3):173-179
7. Buravkova L, Romanor Y, Rykova.M, et al. Cell to cell interactions in changed gravity:ground-based and flight experiments [J]. Acta Astronaut,2005,57(2-8):67-74
8. Gruener R, Roberts R, Roistetter R. Reduced receptor aggregation and altered cytoskeleton in cultured myocytes after spaceflight[J]. Biol Sci Space, 1994,8(2):79-93
9. Schatten H, Lewis ML, Chakrabarti A. Spaceflight and clinorotation cause cytoskeleton and mitochondria changes and increases in apoptosis in cultured cells [J]. Acta Astronaut,2001,49(3-10):399-418
1. Infanger.M, Kossmehl.P, Shakibaei.M. Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity[J]:Impact of vascular endothelial growth factor. Apoptosis, 2006,11(5):749-764
2. Infanger.M, Ulbrich.C, Baatout.S. Modeled gravitational unloading induced downregulation of endothelin-1 in human endothelial cells[J]. J Cell Biochem, 2007,101(6):1439-1455
3. Ulbrich.C, Westphal.K, Baatout.S. Effects of basic fibroblast growth factor on endothelial cells under conditional of simulated microgravity[J]. J Cell Biochem, 2008,104(4):1324-1341
4.林跃辉,王敏.PI3K-AKT信号转导途径与凋亡的关系[J] 国际病理科学与临 床杂志,2005,25(4)：307-310
5.王静,李东野,夏勇 转染Akt1基因对缺血再灌注大鼠心肌线粒体通透性转换的影响 中国病理生理杂志,2010,26(1)：80-85
6. Jianhua Feng, Eliana Lucchinetti, Preeti Ahuja, et al. Isoflurane postconditioning prevents opening of the mitochondrial permeability transition pore through inhibition of glycogen synthase kinase 3β. Anesthesiology,2005,103:987-995
7.宋其蔓,孙洞箫细胞凋亡与P21关系的研究 医学综述,2008,14(6)：816-818
8.韩凯,马彦卓,许旭东.内皮素-1对大鼠骨髓内皮前体细胞凋亡和细胞周期的影响[J].第四军医大学学报,2008,29(22)：2026-2028
9. Priya Kulasekaren, Casey A Scavone, David S Rogers, et al. Endothelin-1 and TGF-β independently induce fibroblast resistance to apoptosis via Akt activation[J]. Am J Respir Cell Mol Biol,2009,2(in Press)
10.陈樑 张红锋 杨帆.Akt抗凋亡机制与癌症治疗[J].生命的化学,2004,24(5)：431-432
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