细胞周期蛋白Hcdc14A在高糖和高游离脂肪酸及低氧联合刺激诱导脑血管内皮细胞损伤中的调控机制研究
|
摘要
第一部分
高糖、高游离脂肪酸和低氧刺激对Hcdc 14A和相关周期蛋白表达及细胞增殖和凋亡的影响
目的：观察高糖、高游离脂肪酸(FFA)和低氧刺激对人脑血管内皮细胞Hcdc14A和相关周期蛋白表达及细胞增殖和凋亡的影响。
方法：分别采用不同条件的高糖、高FFA和低氧刺激人脑血管内皮细胞：RT-PCR和Western-blot检测Hcdc14A的mRNA和蛋白表达；Western-blot检测相关周期蛋白cyclinB、cyclinD、cyclinE和P53蛋白表达；XTT法和CCK8法测定细胞增殖率和存活率；流式细胞术和caspase3活性检测法观察细胞凋亡；流式细胞术观察细胞周期；荧光探针观察细胞骨架蛋白F-actin空间结构；电镜观察细胞超微结构。
结果：1．高糖、高FFA和低氧刺激均可明显下调人脑血管内皮细胞Hcdc14A的mRNA和蛋白表达。2．高糖、高FFA和低氧刺激均可下调cyclinB、cyclinD和cyclinE蛋白表达，上调P53蛋白表达。3．高糖、高FFA和低氧刺激均使细胞增殖率和存活率下降。4．高糖、高FFA和低氧刺激均使细胞凋亡率增加，Caspase-3活性增强。5．高糖、高FFA和低氧刺激均明显下调细胞周期中S期和G2-M比例。6．高糖、高FFA和低氧刺激均对细胞骨架蛋白F-actin空间结构产生一定影响。7．电镜观察发现高糖、高FFA和低氧刺激后凋亡细胞增多，联合刺激组凋亡小体形成较明显。
结论：高糖、高FFA和低氧刺激均可明显下调人脑血管内皮细胞Hcdc 14A和相关周期蛋白表达，阻滞细胞周期，降低细胞增殖率，诱导细胞凋亡，并对细胞骨架蛋白F-actin空间结构和细胞超微结构产生一定影响，三者联合刺激上述作用最为明显。
第二部分
过表达Hcdc14A对相关周期蛋白表达及细胞增殖和凋亡的影响
目的：观察过表达Hcdc14A对相关周期蛋白表达及细胞增殖和凋亡的影响。方法：构建Hcdc14A真核表达载体并转染HEK293细胞和Hela细胞；共聚焦显微镜观察Hcdc14A在细胞分裂期的定位；Western-blot检测重组质粒转染后Hcdc14A表达效率及其过表达对cyclinB、cyclinD、cyclinE和P53蛋白表达的影响；CCK8方法检测重组质粒转染并给予高糖、高FFA和低氧联合刺激后的细胞存活率；流式细胞术和caspase3活性测定法观察转染和联合刺激后的细胞凋亡；流式细胞术观察转染和联合刺激后的细胞周期；荧光探针观察转染和联合刺激后细胞骨架蛋白F-actin空间结构；电镜观察转染和联合刺激后的细胞超微结构。
结果：1．成功构建并转染Hcdc14A真核表达载体(pEGFP-C2-Hcdc14A质粒)，重组质粒转染后Hcdc 14A表达显著上调，发现其在细胞分裂期定位于纺锤体两极和中心体上。2．重组质粒转染后Hcdc14A表达显著上调，同时cyclinB、cyclinD、cyclinE和P53蛋白表达均不同程度上调。3．转染后细胞增殖率明显增加。4．转染后caspase3活性有所增强。5．转染后细胞周期S期和G2-M比例明显增加。6．转染后细胞骨架蛋白F-actin出现解聚样改变。7．电镜观察发现转染后少部分细胞呈现肿瘤化改变。
结论：过表达Hcdc14A可同时上调cyclinB、cyclinD、cyclinE和P53蛋白表达，加速细胞周期进程，提高细胞增殖率，促进细胞骨架蛋白F-actin解聚，同时也可能影响细胞凋亡进程。提示过表达Hcdc14A可同时影响相关周期蛋白表达和细胞周期进程，进而影响细胞的增殖和凋亡进程。
第三部分
siRNA干扰Hcdc14A对相关周期蛋白表达及细胞增殖和凋亡的影响
目的：观察siRNA干扰Hcdc14A基因表达对相关周期蛋白表达及细胞增殖和凋亡的影响。
方法：设计合成3对靶向Hcdc14A特定序列的siRNA并转染细胞：RT-PCR和Western-blot验证Hcdc14A表达；Western-blot检测siRNA干扰对cyclinB、cyclinD、cyclinE和P53蛋白表达的影响；CCK8方法检测siRNA干扰后的细胞存活率；caspase2活性测定法观察siRNA干扰后的细胞凋亡；流式细胞术观察siRNA干扰后的细胞周期；荧光探针观察siRNA干扰后的细胞骨架蛋白F-actin空间结构；电镜观察siRNA干扰后的细胞超微结构。
结果：1．siRNA能显著下调Hcdc14A的mRNA和蛋白表达水平。2．siRNA干扰还可同时下调cyclinB和cyclinD蛋白表达，上调P53蛋白表达，对cyclinE无明显影响。3．siRNA干扰后细胞存活率明显下降。4．siRNA干扰后细胞凋亡明显增加。5．siRNA干扰后细胞周期中S期和G2-M比例明显减少。
6．siRNA干扰后细胞骨架蛋白F-actin呈现一定聚合样改变。7．电镜观察发现siRNA干扰后凋亡细胞增多。
结论：siRNA干扰Hcdc14A基因表达的同时还可影响相关周期蛋白cyclinB、cyclinD和P53的表达，阻滞细胞周期进程，抑制细胞增殖，诱导细胞凋亡，并可能对骨架蛋白F-actin空间结构产生一定影响。提示下调Hcdc14A基因表达可能引起整个细胞周期调控网络功能紊乱，导致细胞正常生理功能发生改变。
第四部分
G-CSF对Hcdc14A和相关周期蛋白表达的影响及其细胞保护作用机制
目的：观察G-CSF对人脑血管内皮细胞Hcdc14A和相关周期蛋白表达的影响，初步探讨G-CSF对联合刺激后人脑血管内皮细胞的保护作用及其机制。
方法：不同条件G-CSF刺激人脑血管内皮细胞，CCK8法和caspase3活性测定法观察G-CSF对细胞增殖和凋亡的影响；XTT法和CCK8法检测G-CSF预处理不同时间对高糖、高FFA和低氧联合刺激后细胞增殖率和存活率的影响；Western-blot检测G-CSF预处理对Hcdc14A及相关周期蛋白表达的影响；流式细胞术和caspase3活性测定法观察G-CSF预处理对细胞凋亡的影响；流式细胞术检测G-CSF预处理对细胞周期的影响；荧光探针观察G-CSF预处理对细胞骨架蛋白F-actin空间结构的影响：电镜观察G-CSF预处理对细胞形态学的影响；荧光探针检测G-CSF预处理对活性氧(ROS)、一氧化氮(NO)、内皮型一氧化氮合成酶(eNOS)、钙离子(Ca~(2+))及线粒体膜电位(MMP)等氧化应激相关指标的影响；ELISA和免疫荧光等方法检测G-CSF预处理对PI3K／AKT和MAPK信号通路的影响。
结果：1．G-CSF刺激可促进细胞增殖并抑制细胞凋亡。2．G-CSF预处理12h可明显提高联合刺激后的细胞增殖率和存活率。3．G-CSF能够明显上调Hcdc14A、cyclinB和cyelinE蛋白表达，下调P53蛋白表达，对cyclinD无明显影响。4．G-CSF预处理可明显抑制联合刺激诱导的细胞凋亡。5．G-CSF预处理可上调S期和G2-M期细胞比例。6．G-CSF预处理可促进细胞骨架蛋白F-actin解聚。7．电镜观察发现G-CSF预处理后凋亡细胞有所减少。8．G-CSF预处理可明显拮抗联合刺激诱导的氧化应激，上调NO、eNOS和MMP水平，降低ROS和Ca~(2+)水平。9．G-CSF可激活PI3K／AKT和ERK1／2信号通路，并抑制JNK和p38信号通路。
结论：G-CSF能够上调Hcdc14A和相关周期蛋白cyclinB、cyclinE蛋白表达，下调P53蛋白表达，调控细胞周期进程，促进细胞增殖，抑制细胞凋亡，拮抗高糖、高FFA和低氧联合刺激诱导的氧化应激，并可激活PI3K／AKT和ERK1／2信号通路，抑制JNK和p38信号通路。提示G-CSF可能是通过调控Hcdc14A和相关周期蛋白表达、拮抗氧化应激、影响PI3K／AKT和MAPK信号通路等多种机制发挥其内皮细胞保护作用。
PartⅠ
The influences of different stimuli on the expressions ofHcdc14A and its related cyclins,cell proliferation andapoptosis in HBVEC
OBJECTIVE:To investigate the influence of high glucose,high FFA and hypoxiaon protein Hcdcl4A,related cyclins,cell proliferation and apoptosis in HBVEC.
METHODS:HBVECs were treated with high glucose,high FFA and hypoxia.Theexpression of Hcdcl4A at the mRNA and protein level was detected by RT-PCR andWestern Blot,respectively.At the same time,the expressions of cyclinB,cyclinD,cyclinE and P53 were detected by Western Blot.The cell proliferation ability andsurvival ability of HBVECs which were treated by above conditions were furtherconfirmed by XTT and CCK8.The cell apoptosis was detected by AnnexinV/PI andcaspase3 activity,the cell cycle by flow cytometry,the spatial structure ofcytoskeletal protein by fluorescent probe,and cell ultrastructure by electronmicroscope.
RESULTS:1.The expression of mRNA and protein of Hcdcl4A were decreased inHBVECs,as stimulated by high glucose,high FFA and hypoxia in different ways,with the most obvious decrease of Hcdcl4A existing in the combined intervention ofabove stimuli.2.The expression of protein cyclinB,cyclinD and cyclinE weredecreased,and P53 was increased after different stimuli.3.The proliferation abilityand survival ability of HBVECs were reduced after being stimulated by aboveconditions.4.The cell apoptosis was increased and the caspase3 activity was raised.5.The cell proportion of S phase and G2-M phase after above stimulations were lower than that of control.6.The spatial structure of cytoskeletal protein waschanged after above stimulations.7.The apoptosis cell and apoptotic body wereobviously increased after stimulations under electron microscope.CONCLUSIONS:The expression of protein Hcdc 14A was decreased in cells treatedwith high glucose,high FFA and hypoxia in different ways,and related cyclins werealso influenced.The cell cycle was partly blocked,cytoskeletal protein and cellultrastructure were interfered or destroyed,the proliferation ability and survival abilityof HBVECs were reduced,and the cell apoptosis was increased after being treated byabove conditions.
PartⅡ
The influence of overexpressing of Hcdcl4A on relatedcyclins,cell proliferation and apoptosis
OBJECTIVE:To investigate the influence of overexpressing Hcdc14A on relatedcyclins,cell proliferation and cell apoptosis.
METHODS:The full-length cDNA sequence was subcloned into green fluorescencerotein vector pEGFP to study the location of Hcdcl4A in the HEK293 cells and Helaells by confocal microscope.We also tried to detect the expressions of cyclinB,cyclinD,cyclinE and P53 by Western Blot when Hcdcl4A was overexpressed.Theproliferation ability of HBVECs was further confirmed by CCK8,the cell apoptosiswas detected by AnnexinV/PI and caspase3 activity respectively,the cell cycle by flow cytometry,the spatial structure of cytoskeletal protein by fluorescent probe,andcell ultrastructure by electron microscope after Hcdc 14A was overexpressed.
RESULTS:1.Hcdcl4A was located on the two poles of spindle apparatus andcellular centrosome.2.Not only the expression of protein Hcdcl4A was strikinglyincreased,but also cyclinB,cyclinD,cyclinE and P53 were all increased afterHcdcl4A was overexpressed.3.The cell proliferation ability was increased afterHcdc 14A was overexpressed.4.The cell apoptosis was not obviously increased,butthe caspase3 activity was obviously raised after Hcdcl4A was overexpressed.5.Thecell proportions of S phase and G2-M phase after Hcdcl 4A was overexpressed wereraised.6.The cytoskeletal protein were obviously depolymerized after Hcdcl 4A wasoverexpressed.7.There were some tumor-like cell found under electron microscopeafter Hcdcl4A was overexpressed.
CONCLUSIONS:The overexpression of protein Hcdcl4A could up-regulated theexpression of cyclinB,cyclinD,cyclinE and P53,accelerate cell cycle,promote cellproliferation,induced depolymerization of cytoskeletal protein,and perhaps disturbthe normal apoptosis proceeding.Thus these results suggested that the interactionbetween Hcdcl4A and cellular cyclical proteins after Hcdcl4A was overexpressedmight play a role in cell proliferation and apoptosis.
OBJECTIVE:To investigate the influence of knockdown of Hcdcl4A with siRNAon related cyclins,cell proliferation and cell apoptosis.
METHODS:To synthesize three pairs siRNA targeting definite sequence ofHcdcl4A and transfect HEK293 cell.After the total RNA and protein was extractedfrom these treated cells,the expressions of RNA and protein of Hcdcl4A weredetected by RT-PCR and Westem Blot respectively to verify the change of Hcdcl 4Ain the HEK293 cells.We also tried to detect the expressions of cyclinB,cyclinD,cyclinE and P53 by Westem Blot after siRNA transfection.The cell proliferationability of HBVECs was confirmed by CCK8,the cell apoptosis was detected bycaspase3 activity,the cell cycle by flow cytometry,the spatial structure ofcytoskeletal protein by fluorescent probe,and cell ultrastructure by electronmicroscope after siRNA transfection.
RESULTS:1.Not only the expression of protein Hcdcl4A was strikinglydown-regulated,but also that of cyclinB and cyclinD were all decreased and that ofP53 was increased after siRNA transfection.2.The cell proliferation ability wasdecreased compared with that of control after siRNA transfection.3.The cellapoptosis was obviously increased compared with that of control after siRNAtransfection.4.The cell proportions of S phase and G2-M phase after siRNAtransfection were decreased compared with that of control.5.The cytoskeletalprotein became polymerized and obscure after siRNA transfection.6.The apoptosiscell and apoptotic body were obviously increased after siRNA transfection.
CONCLUSIONS:The knockdown of Hcdcl4A could also affect the expression ofcyclinB,cyclinD and P53,block cell cycle,promote polymerization of cytoskeletalprotein,suppress cell proliferation,and induce cell apoptosis.Thus these resultssuggested that knockdown of Hcdcl4A cause functional disorder of cell cycleregulation network and disturb the cell normal physiological function.
PartⅣ
The influence of G-CSF on Hcdcl4A and its related cyclinsand the mechanism of G-CSF cytoprotective action onHBVEC
OBJECTIVE:To detect the influence of G-CSF on Hcdcl4A and its related cyclins,and investigate G-CSF cytoprotection mechanism on HBVEC after combinedintervention.
METHODS:After HBVECs treated by G-CSF in different ways,the proliferationability and cell apoptosis were confirmed by CCK and caspase3 activity respectively.After different G-CSF pretreatment,the cell proliferation ability and survival abilityof HBVECs stimulated with high glucose,high FFA and hypoxia were examined byXTT and CCK8.After G-CSF pretreatment the apoptosis of HBVECs treated withabove combined stimulation was confirmed with AnnexinV/PI and caspase3activity.We also tried to detect the expressions of Hcdcl4A,cyclinB,cyclinD,cyclinE and P53 by Westem Blot after combined stimulation with G-CSFpretreatment.After combined stimulation with G-CSF pretreatment,the cell cyclewas detected by flow cytometry,the cytoskeletal protein by fluorescent probe,cellultrastructure by electron microscope,oxidative stress related indexes including ROS,NO,eNOS,Ca~(2+)and MMP by fluorescent probe,and PI3K/AKT and MAPK signalpassway by immunofluorescence.
RESULTS:1.G-CSF(100nM for 72h)displayed obvious contribution on promotingproliferation and suppressing apoptosis.2.G-CSF pretreatment for 12h or 24h hadthe similar effects on raising cell proliferation and decreasing cell apoptosis(P＞0.05).3.Not only the expressions of protein Hcdcl4A,cyclinB and cyclinE were strikinglyincreased,but also that of P53 was decreased after combined stimulation withG-CSF pretreatment.4.After combined stimulation with G-CSF pretreatment, theaflavinehe cell proportions of S phase and G2-M phase were raised,thecytoskeletal protein was obviously depolymerized,and the apoptosis cell andapoptotic body were obviously decreased.5.G-CSF pretreatment could relieve theoxidative stress by elevating NO,eNOS and MMP,degrading ROS and Ca~(2+).6.G-CSF pretreatment could lessen the damage on HBVECs induced with combinedstimulation by refreshing AKT and ERK1/2 and blocking JNK and p38 passways.
CONCLUSIONS:G-CSF could up-regulate the expressions of Hcdcl4A,cyclinB,and cyclinE,and down-regulate the expression of P53,regulate cell cycle,promotecell proliferation,suppress cell apoptosis,reduce oxidiative stress in HBVECsinduced by high glucose,high FFA and hypoxia.G-CSF perhaps producedcytoprotection and anti-apoptotic effect through regulating PI3K/Akt and MAPKpathways.
高糖、高游离脂肪酸和低氧刺激对Hcdc 14A和相关周期蛋白表达及细胞增殖和凋亡的影响
目的：观察高糖、高游离脂肪酸(FFA)和低氧刺激对人脑血管内皮细胞Hcdc14A和相关周期蛋白表达及细胞增殖和凋亡的影响。
方法：分别采用不同条件的高糖、高FFA和低氧刺激人脑血管内皮细胞：RT-PCR和Western-blot检测Hcdc14A的mRNA和蛋白表达；Western-blot检测相关周期蛋白cyclinB、cyclinD、cyclinE和P53蛋白表达；XTT法和CCK8法测定细胞增殖率和存活率；流式细胞术和caspase3活性检测法观察细胞凋亡；流式细胞术观察细胞周期；荧光探针观察细胞骨架蛋白F-actin空间结构；电镜观察细胞超微结构。
结果：1．高糖、高FFA和低氧刺激均可明显下调人脑血管内皮细胞Hcdc14A的mRNA和蛋白表达。2．高糖、高FFA和低氧刺激均可下调cyclinB、cyclinD和cyclinE蛋白表达，上调P53蛋白表达。3．高糖、高FFA和低氧刺激均使细胞增殖率和存活率下降。4．高糖、高FFA和低氧刺激均使细胞凋亡率增加，Caspase-3活性增强。5．高糖、高FFA和低氧刺激均明显下调细胞周期中S期和G2-M比例。6．高糖、高FFA和低氧刺激均对细胞骨架蛋白F-actin空间结构产生一定影响。7．电镜观察发现高糖、高FFA和低氧刺激后凋亡细胞增多，联合刺激组凋亡小体形成较明显。
结论：高糖、高FFA和低氧刺激均可明显下调人脑血管内皮细胞Hcdc 14A和相关周期蛋白表达，阻滞细胞周期，降低细胞增殖率，诱导细胞凋亡，并对细胞骨架蛋白F-actin空间结构和细胞超微结构产生一定影响，三者联合刺激上述作用最为明显。
第二部分
过表达Hcdc14A对相关周期蛋白表达及细胞增殖和凋亡的影响
目的：观察过表达Hcdc14A对相关周期蛋白表达及细胞增殖和凋亡的影响。方法：构建Hcdc14A真核表达载体并转染HEK293细胞和Hela细胞；共聚焦显微镜观察Hcdc14A在细胞分裂期的定位；Western-blot检测重组质粒转染后Hcdc14A表达效率及其过表达对cyclinB、cyclinD、cyclinE和P53蛋白表达的影响；CCK8方法检测重组质粒转染并给予高糖、高FFA和低氧联合刺激后的细胞存活率；流式细胞术和caspase3活性测定法观察转染和联合刺激后的细胞凋亡；流式细胞术观察转染和联合刺激后的细胞周期；荧光探针观察转染和联合刺激后细胞骨架蛋白F-actin空间结构；电镜观察转染和联合刺激后的细胞超微结构。
结果：1．成功构建并转染Hcdc14A真核表达载体(pEGFP-C2-Hcdc14A质粒)，重组质粒转染后Hcdc 14A表达显著上调，发现其在细胞分裂期定位于纺锤体两极和中心体上。2．重组质粒转染后Hcdc14A表达显著上调，同时cyclinB、cyclinD、cyclinE和P53蛋白表达均不同程度上调。3．转染后细胞增殖率明显增加。4．转染后caspase3活性有所增强。5．转染后细胞周期S期和G2-M比例明显增加。6．转染后细胞骨架蛋白F-actin出现解聚样改变。7．电镜观察发现转染后少部分细胞呈现肿瘤化改变。
结论：过表达Hcdc14A可同时上调cyclinB、cyclinD、cyclinE和P53蛋白表达，加速细胞周期进程，提高细胞增殖率，促进细胞骨架蛋白F-actin解聚，同时也可能影响细胞凋亡进程。提示过表达Hcdc14A可同时影响相关周期蛋白表达和细胞周期进程，进而影响细胞的增殖和凋亡进程。
第三部分
siRNA干扰Hcdc14A对相关周期蛋白表达及细胞增殖和凋亡的影响
目的：观察siRNA干扰Hcdc14A基因表达对相关周期蛋白表达及细胞增殖和凋亡的影响。
方法：设计合成3对靶向Hcdc14A特定序列的siRNA并转染细胞：RT-PCR和Western-blot验证Hcdc14A表达；Western-blot检测siRNA干扰对cyclinB、cyclinD、cyclinE和P53蛋白表达的影响；CCK8方法检测siRNA干扰后的细胞存活率；caspase2活性测定法观察siRNA干扰后的细胞凋亡；流式细胞术观察siRNA干扰后的细胞周期；荧光探针观察siRNA干扰后的细胞骨架蛋白F-actin空间结构；电镜观察siRNA干扰后的细胞超微结构。
结果：1．siRNA能显著下调Hcdc14A的mRNA和蛋白表达水平。2．siRNA干扰还可同时下调cyclinB和cyclinD蛋白表达，上调P53蛋白表达，对cyclinE无明显影响。3．siRNA干扰后细胞存活率明显下降。4．siRNA干扰后细胞凋亡明显增加。5．siRNA干扰后细胞周期中S期和G2-M比例明显减少。
6．siRNA干扰后细胞骨架蛋白F-actin呈现一定聚合样改变。7．电镜观察发现siRNA干扰后凋亡细胞增多。
结论：siRNA干扰Hcdc14A基因表达的同时还可影响相关周期蛋白cyclinB、cyclinD和P53的表达，阻滞细胞周期进程，抑制细胞增殖，诱导细胞凋亡，并可能对骨架蛋白F-actin空间结构产生一定影响。提示下调Hcdc14A基因表达可能引起整个细胞周期调控网络功能紊乱，导致细胞正常生理功能发生改变。
第四部分
G-CSF对Hcdc14A和相关周期蛋白表达的影响及其细胞保护作用机制
目的：观察G-CSF对人脑血管内皮细胞Hcdc14A和相关周期蛋白表达的影响，初步探讨G-CSF对联合刺激后人脑血管内皮细胞的保护作用及其机制。
方法：不同条件G-CSF刺激人脑血管内皮细胞，CCK8法和caspase3活性测定法观察G-CSF对细胞增殖和凋亡的影响；XTT法和CCK8法检测G-CSF预处理不同时间对高糖、高FFA和低氧联合刺激后细胞增殖率和存活率的影响；Western-blot检测G-CSF预处理对Hcdc14A及相关周期蛋白表达的影响；流式细胞术和caspase3活性测定法观察G-CSF预处理对细胞凋亡的影响；流式细胞术检测G-CSF预处理对细胞周期的影响；荧光探针观察G-CSF预处理对细胞骨架蛋白F-actin空间结构的影响：电镜观察G-CSF预处理对细胞形态学的影响；荧光探针检测G-CSF预处理对活性氧(ROS)、一氧化氮(NO)、内皮型一氧化氮合成酶(eNOS)、钙离子(Ca~(2+))及线粒体膜电位(MMP)等氧化应激相关指标的影响；ELISA和免疫荧光等方法检测G-CSF预处理对PI3K／AKT和MAPK信号通路的影响。
结果：1．G-CSF刺激可促进细胞增殖并抑制细胞凋亡。2．G-CSF预处理12h可明显提高联合刺激后的细胞增殖率和存活率。3．G-CSF能够明显上调Hcdc14A、cyclinB和cyelinE蛋白表达，下调P53蛋白表达，对cyclinD无明显影响。4．G-CSF预处理可明显抑制联合刺激诱导的细胞凋亡。5．G-CSF预处理可上调S期和G2-M期细胞比例。6．G-CSF预处理可促进细胞骨架蛋白F-actin解聚。7．电镜观察发现G-CSF预处理后凋亡细胞有所减少。8．G-CSF预处理可明显拮抗联合刺激诱导的氧化应激，上调NO、eNOS和MMP水平，降低ROS和Ca~(2+)水平。9．G-CSF可激活PI3K／AKT和ERK1／2信号通路，并抑制JNK和p38信号通路。
结论：G-CSF能够上调Hcdc14A和相关周期蛋白cyclinB、cyclinE蛋白表达，下调P53蛋白表达，调控细胞周期进程，促进细胞增殖，抑制细胞凋亡，拮抗高糖、高FFA和低氧联合刺激诱导的氧化应激，并可激活PI3K／AKT和ERK1／2信号通路，抑制JNK和p38信号通路。提示G-CSF可能是通过调控Hcdc14A和相关周期蛋白表达、拮抗氧化应激、影响PI3K／AKT和MAPK信号通路等多种机制发挥其内皮细胞保护作用。
PartⅠ
The influences of different stimuli on the expressions ofHcdc14A and its related cyclins,cell proliferation andapoptosis in HBVEC
OBJECTIVE:To investigate the influence of high glucose,high FFA and hypoxiaon protein Hcdcl4A,related cyclins,cell proliferation and apoptosis in HBVEC.
METHODS:HBVECs were treated with high glucose,high FFA and hypoxia.Theexpression of Hcdcl4A at the mRNA and protein level was detected by RT-PCR andWestern Blot,respectively.At the same time,the expressions of cyclinB,cyclinD,cyclinE and P53 were detected by Western Blot.The cell proliferation ability andsurvival ability of HBVECs which were treated by above conditions were furtherconfirmed by XTT and CCK8.The cell apoptosis was detected by AnnexinV/PI andcaspase3 activity,the cell cycle by flow cytometry,the spatial structure ofcytoskeletal protein by fluorescent probe,and cell ultrastructure by electronmicroscope.
RESULTS:1.The expression of mRNA and protein of Hcdcl4A were decreased inHBVECs,as stimulated by high glucose,high FFA and hypoxia in different ways,with the most obvious decrease of Hcdcl4A existing in the combined intervention ofabove stimuli.2.The expression of protein cyclinB,cyclinD and cyclinE weredecreased,and P53 was increased after different stimuli.3.The proliferation abilityand survival ability of HBVECs were reduced after being stimulated by aboveconditions.4.The cell apoptosis was increased and the caspase3 activity was raised.5.The cell proportion of S phase and G2-M phase after above stimulations were lower than that of control.6.The spatial structure of cytoskeletal protein waschanged after above stimulations.7.The apoptosis cell and apoptotic body wereobviously increased after stimulations under electron microscope.CONCLUSIONS:The expression of protein Hcdc 14A was decreased in cells treatedwith high glucose,high FFA and hypoxia in different ways,and related cyclins werealso influenced.The cell cycle was partly blocked,cytoskeletal protein and cellultrastructure were interfered or destroyed,the proliferation ability and survival abilityof HBVECs were reduced,and the cell apoptosis was increased after being treated byabove conditions.
PartⅡ
The influence of overexpressing of Hcdcl4A on relatedcyclins,cell proliferation and apoptosis
OBJECTIVE:To investigate the influence of overexpressing Hcdc14A on relatedcyclins,cell proliferation and cell apoptosis.
METHODS:The full-length cDNA sequence was subcloned into green fluorescencerotein vector pEGFP to study the location of Hcdcl4A in the HEK293 cells and Helaells by confocal microscope.We also tried to detect the expressions of cyclinB,cyclinD,cyclinE and P53 by Western Blot when Hcdcl4A was overexpressed.Theproliferation ability of HBVECs was further confirmed by CCK8,the cell apoptosiswas detected by AnnexinV/PI and caspase3 activity respectively,the cell cycle by flow cytometry,the spatial structure of cytoskeletal protein by fluorescent probe,andcell ultrastructure by electron microscope after Hcdc 14A was overexpressed.
RESULTS:1.Hcdcl4A was located on the two poles of spindle apparatus andcellular centrosome.2.Not only the expression of protein Hcdcl4A was strikinglyincreased,but also cyclinB,cyclinD,cyclinE and P53 were all increased afterHcdcl4A was overexpressed.3.The cell proliferation ability was increased afterHcdc 14A was overexpressed.4.The cell apoptosis was not obviously increased,butthe caspase3 activity was obviously raised after Hcdcl4A was overexpressed.5.Thecell proportions of S phase and G2-M phase after Hcdcl 4A was overexpressed wereraised.6.The cytoskeletal protein were obviously depolymerized after Hcdcl 4A wasoverexpressed.7.There were some tumor-like cell found under electron microscopeafter Hcdcl4A was overexpressed.
CONCLUSIONS:The overexpression of protein Hcdcl4A could up-regulated theexpression of cyclinB,cyclinD,cyclinE and P53,accelerate cell cycle,promote cellproliferation,induced depolymerization of cytoskeletal protein,and perhaps disturbthe normal apoptosis proceeding.Thus these results suggested that the interactionbetween Hcdcl4A and cellular cyclical proteins after Hcdcl4A was overexpressedmight play a role in cell proliferation and apoptosis.
OBJECTIVE:To investigate the influence of knockdown of Hcdcl4A with siRNAon related cyclins,cell proliferation and cell apoptosis.
METHODS:To synthesize three pairs siRNA targeting definite sequence ofHcdcl4A and transfect HEK293 cell.After the total RNA and protein was extractedfrom these treated cells,the expressions of RNA and protein of Hcdcl4A weredetected by RT-PCR and Westem Blot respectively to verify the change of Hcdcl 4Ain the HEK293 cells.We also tried to detect the expressions of cyclinB,cyclinD,cyclinE and P53 by Westem Blot after siRNA transfection.The cell proliferationability of HBVECs was confirmed by CCK8,the cell apoptosis was detected bycaspase3 activity,the cell cycle by flow cytometry,the spatial structure ofcytoskeletal protein by fluorescent probe,and cell ultrastructure by electronmicroscope after siRNA transfection.
RESULTS:1.Not only the expression of protein Hcdcl4A was strikinglydown-regulated,but also that of cyclinB and cyclinD were all decreased and that ofP53 was increased after siRNA transfection.2.The cell proliferation ability wasdecreased compared with that of control after siRNA transfection.3.The cellapoptosis was obviously increased compared with that of control after siRNAtransfection.4.The cell proportions of S phase and G2-M phase after siRNAtransfection were decreased compared with that of control.5.The cytoskeletalprotein became polymerized and obscure after siRNA transfection.6.The apoptosiscell and apoptotic body were obviously increased after siRNA transfection.
CONCLUSIONS:The knockdown of Hcdcl4A could also affect the expression ofcyclinB,cyclinD and P53,block cell cycle,promote polymerization of cytoskeletalprotein,suppress cell proliferation,and induce cell apoptosis.Thus these resultssuggested that knockdown of Hcdcl4A cause functional disorder of cell cycleregulation network and disturb the cell normal physiological function.
PartⅣ
The influence of G-CSF on Hcdcl4A and its related cyclinsand the mechanism of G-CSF cytoprotective action onHBVEC
OBJECTIVE:To detect the influence of G-CSF on Hcdcl4A and its related cyclins,and investigate G-CSF cytoprotection mechanism on HBVEC after combinedintervention.
METHODS:After HBVECs treated by G-CSF in different ways,the proliferationability and cell apoptosis were confirmed by CCK and caspase3 activity respectively.After different G-CSF pretreatment,the cell proliferation ability and survival abilityof HBVECs stimulated with high glucose,high FFA and hypoxia were examined byXTT and CCK8.After G-CSF pretreatment the apoptosis of HBVECs treated withabove combined stimulation was confirmed with AnnexinV/PI and caspase3activity.We also tried to detect the expressions of Hcdcl4A,cyclinB,cyclinD,cyclinE and P53 by Westem Blot after combined stimulation with G-CSFpretreatment.After combined stimulation with G-CSF pretreatment,the cell cyclewas detected by flow cytometry,the cytoskeletal protein by fluorescent probe,cellultrastructure by electron microscope,oxidative stress related indexes including ROS,NO,eNOS,Ca~(2+)and MMP by fluorescent probe,and PI3K/AKT and MAPK signalpassway by immunofluorescence.
RESULTS:1.G-CSF(100nM for 72h)displayed obvious contribution on promotingproliferation and suppressing apoptosis.2.G-CSF pretreatment for 12h or 24h hadthe similar effects on raising cell proliferation and decreasing cell apoptosis(P＞0.05).3.Not only the expressions of protein Hcdcl4A,cyclinB and cyclinE were strikinglyincreased,but also that of P53 was decreased after combined stimulation withG-CSF pretreatment.4.After combined stimulation with G-CSF pretreatment, theaflavinehe cell proportions of S phase and G2-M phase were raised,thecytoskeletal protein was obviously depolymerized,and the apoptosis cell andapoptotic body were obviously decreased.5.G-CSF pretreatment could relieve theoxidative stress by elevating NO,eNOS and MMP,degrading ROS and Ca~(2+).6.G-CSF pretreatment could lessen the damage on HBVECs induced with combinedstimulation by refreshing AKT and ERK1/2 and blocking JNK and p38 passways.
CONCLUSIONS:G-CSF could up-regulate the expressions of Hcdcl4A,cyclinB,and cyclinE,and down-regulate the expression of P53,regulate cell cycle,promotecell proliferation,suppress cell apoptosis,reduce oxidiative stress in HBVECsinduced by high glucose,high FFA and hypoxia.G-CSF perhaps producedcytoprotection and anti-apoptotic effect through regulating PI3K/Akt and MAPKpathways.
引文
1.Marcel Doree,Tim Hunt.From Cdc2 to Cdkl:when did the cell cycle kinase join its cyclin partner[J].Journal of Cell Science,2002;115(12):2461-2464.
2.Nasmyth K.Viewpoint:putting the cell cycle in order[J].Science,1996;274(5293):1640-1645.
3.Morgan DO.Cyclin-dependent kinases:engines,clocks,and microprocessors[J].Annu Rev Cell Dev Biol,1997;13(11):261-291.
4.Willamson J,Chang K,Frangos M,et al.Hyperglycemic pseudohypoxia and diabetic complications[J].Diabetes,1993;42(6):801-813.
5.Visintm R,Craig K,Hwang ES,et al.The phosphatase Cdcl4 triggers mitotic exit by reversal of Cdk-dependent phosphorylation [J]. Mol Cell, 1998;2(6): 709-71.
6. Wan J, Xu H, Grunstein M.Cdcl4 of saccharomyces cerevisiae. Cloning, sequence analysis and transcription during the cell cycle [J]. J Biol Chem, 1992; 267(16): 11274-11280.
7. Li L, Ernsting BR, Wishart MJ, et al. A family of putative tumor suppressors is structurally and functionally conserved in humans and yeast [J]. J Biol Chem, 1997;272(47): 29403-29406.
8. Hyekyung P Cho, Yie Liu, Maria Gomez, et al. The dual-specificity phosphatase cdcl4B bundles and stabilizes microtubules[J]. Molecular and Cellular Biology, 2005; 25(1): 4541-4551.
9. Grzegorz Nalepa, J. Wade Harper. Visualization of a highly organized intranuclear network of filament in living mammalian cells [J]. Cell Motility and Cytoskeleton, 2004; 59(2): 94-108.
10. Brett KK, Zachary AZ, Harry C,et al. Disruption of centrosome structure, chromosome segregation, and cytokinesis by misexpression of human Cdcl4A phosphatase [J]. Molecular Biology of the Cell, 2002; 13(7): 2289-2300.
11. Niels Mailand, Claudia Lukas, Brett K, et al. Deregulated human Cdcl4A phosphatase disrupts centrosome separation and chromosome segregation[J]. Nature Cell Biology, 2002; 4(4): 317-322.
12. Vazquez-Novelle MD, Esteban V, Bueno A,et al.Functional homology among human and fission yeast cdcl4 phophatases[J]. J Biol Chem, 2005; 280(32): 29144-29150.
1.Cugnet-Anceau C,Bauduceau B.Glycaemic control and cardiovascular morbi-mortality:The contribution of the 2008 studies[J].Ann Endocrinol(Paris),2009;70(1):48-54.
2.Kissela BM,Khoury J,Kleindorfer D,et al.Epidemiology of ischaemic stroke in patients with diabetes:the greater Cincinnati/northern Kentucky Stroke Study[J].Diabetes Care,2005;28(2):355-359.
3.Ahmad FK,He Z,King GL.Molecular targets of diabetic cardiovascular complications[J].Curr Drug Target,2005;6(4):487-494.
4.De Caterina R.Endothelial dysfunctions:common denominatorsin vascular disease[J].Curr Opin Clin Nutr Metab Care.2000;3(6):453-467.
5.Szrnitko PE,Wang CH,Weisel RD,et al.Biomarkers of vascular disease linking inflammation to endothelial activation:Part Ⅱ[J].Circulation,2003;108(1):2041-2058.
6.Verma S,Buchanan MR,Anderson TJ.Endothelial function testing as a biomarker of vascular disease[J].Circulation,2003;108(1):2054-2069.
7.Visintin R,Craig K,Hwang ES,et al.The phosphatase Cdcl4 triggers mitotic exit by reversal of Cdk-dependent phosphorylation[J].Mol Cell,1998;2(6):709-718.
8.MICHIELS C.Endothelial cell functions[J].J Cell Physiol,2003;196 (3):430-443.
9.Nagy Z,VastagM,Skopal J,et al.Human brain microvessel endothelial cell culture as a model system to study vascular factors ofischemic brain[J].Keio-J-Med,1996;45(3):200-206.
10.D'Agnillo F,Wood F,Porras C,et al.Effects ofhypoxia and glutathione depletion on hemoglobin and myoglobin-mediated oxidative stress toward endothelium[J].Bichim Biophys Acta,2000;1495(2):150-159.
11.Samarasinghe DA,Tapner M,Ferrell GC.Role of oxidative stress on hypoxia-reoxygenation injury to cultured rat hepatic sinusoidal endothelial cells[J].Hepatology,2000;31(1):160-165.
12.Shostak HDC,Lemasters JJ,Edgell CJ,et al.Role oflCElike proteases in endothelial cell hypoxic and reperfusion injury[J].BiochemBiophys ResCommun,1997;231(3):844-847.
13.Faller DV.Endothelial cell responses to hypoxic stress[J].Clin Exp Pharmacol Physiol,1999;26(1):74-84.
14.Brett KK,Zachary AZ,Harry C,et al.Disruption of centrosorre structure,chromosome segregation,and cytokinesis by misexpression of human Cdcl4A phosphatase[J].Molecular Biology of the Cell,2002;13(7):2289-2300.
15.Vazquez-Novelle MD,Esteban V,Bueno A,et al.Functional homology among human and fission yeast cdcl4 phophatases[J].J Biol Che,2005;280(32):29144-29150.
16.Fields S,Song O-K A novel genetic system to detect protein-protein interactions[J].Nature,1989;340(6230):245-246.
17.Golemis E.Protein-protein interactions,A Molecular Cloning Manual[M].New York:Cold Spring Harbour Laboratory press, 2002:1-682.
18. Bartel PL, Fields S. The yeast two-hybrid system [M]. New York: Oxford University Press, 1997:425-428.
19. Sandrock B, Tirode F, Egly JM. Methods in Molecular Biology: Two-Hybrid System: Methods and Protocols[M]. New Jersey: Human press, 2001:271-289.
20. Dawai T,Matsumoto M,Takeda K,et al.ZIP kinase a novel serine/threonine kinase which mediates apoptosis [J]. Mol Cell Biol, 1998; 18(3): 1642-1651.
21. Kogel D, Reimertz C, Dussmann H, et al. The death associated protein (DAP) kinase homologue Dlk/ZIP kinase induces pl9ARF-and p53-independent apoptosis[J]. Eur J Cance, 2003; 39(2):249-256.
22. Kogel D, Plottner O, Landsberg G et al. Cloning and characterization of Dlk, a novel serine/threonine kinase that is tightly associated with chromatin and phophorylates core histones [J]. Oncogene, 1998; 17(20): 2645-2654.
1.Wolf G Molecular mechanisms of renal hypertrophy:role of p27Kipl[J].Kidney Int,1999;56(4):1262-1265.
2.Brett KK,Zachary AZ,Harry C,et al.Disruption of Centrosome Structure,Chromosome Segregation,and Cytokinesis by Misexpression of Human Cdc14A Phosphatase[J].Molecular Biology of the Cell,2002;13(7):2289-2300.
3.Piel,M,Nordberg,J,Euteneuer,U,Bomens,M.Centrosome-dependent exit of cytokinesis in animal cells[J].Science,2001;291(5508):1550-1553.
4.Hyekyung P Cho,Yie Liu,Maria Gomez,et al.The dual-specificity phosphatase cdc14B bundles and stabilizes microtubules[J].Molecular and Cellular Biology,2005;25(1):4541-4551.
5.Pockwinse SM,Krockmalnic G,Doxsey S J,Nickerson J,et al.Cell cycle independent interaction of CDC2 with the centrosome,which is associated with the nuclear matrix-intermediate filament scaffold[J].Proc Natl Acad Sci,1997;94(7):3022-3027.
6.Okuda M,Hom HF,Tarapore P,et al.Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication[J].Cell,2000;103(1):127-140.
7.Michelle T P,Adrienne M S,Frederick A D,et al.The p53-targeting human phosphatase hCdcl4A interacts with the Cdkl/cyclin B complex and is diffententially expressed in human cancers[J].Molecular Cancer,2006;5(25):2100-2110.
8.Brett K.Kaiser,Zachary A.Zimmerman,Harry Charbonneau,et al.Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdcl4A Phosphatase[J]. Mol Biol Cell, 2002;13(7): 2289-2300.
9. Bembenek J, Yu H. Regulation of the anaphase-promoting complex by the dual specificity phosphatase human Cdcl4a[J].J. Biol. Chem, 2001;276(51): 48237-48242.
10. Brett K. Kaiser, Zachary A. Zimmerman, Harry Charbonneau, et al. Disruption of centrosome structure, chromosome segregation, and cytokinesis by misexpression of human Cdcl4A phosphatase[J]. Molecular Biology of the Cell, 2002;13(7): 2289-2300.
11. Mailand N, Lukas C, Kaiser BK, Jackson PK, Bartek J, Lukas J. Deregulated human Cdcl4A phosphatase disrupts centrosome separation and chromosome segregation [J]. Nat. Cell Biol, 2002;4(4):317-322.
12. M. Dolores Va' zquez-Novelle, Vero' nica Esteban, Avelino Bueno, Man'a P. Sacristan. Functional Homology among Human and Fission Yeast Cdcl4 Phosphatases [J]. J Biol Chem, 2005;280(32): 29144-29150.
13. Martinsson HS ,Starborg M, Erlandsson F. et al. Single cell analysis of Gl check points-the relationship between the restriction point and phosphorylation of pRb [J]. Exp Cell Res, 2005;305(2):383-391.
14. Keyomarsi K, Herliczek TW. The role of Cyclin E in cell proliferation, development and cancer [J]. Prog Cell Cycle Res,1997;3(l): 171-191.
15. Montero S, Guzman C,Vargas C, et al. Prognostic value of cytosolic p53 protein in breast cancer [J]. Tumor Biol, 2001;22 (5): 337-344.
16. Burns TF, El-deiry WS. The P53 pathway and apoptosis[J].J Cell Physiol, 1999;181(2): 231-239.
17. Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage [J]. Science, 1998;282(5393): 1497-1501.
18. Barboule N, Chadebech P, Baldin V, et al. Involvement of p21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line[J].Oncogene, 1997;15(23): 2867-2875.
19. Li L, Ljungman M, Dixon JE. The human Cdcl4 phosphatases interact with and dephosphorylate the tumor suppressor protein p53 [J]. J. Biol. Chem, 2000;275(4): 2410-2414.
20. Lowe SW, Bodis S, Bardeesy N, et al. Apoptosis and the prognostic significance of P53 mutation [J]. Cold Spring Harb Symp Quant Biol, 1994;59: 419-426.
21. Bullock AN, Fersht AR. Rescuing the function of mutant p53 [J]. Nat Rev Cancer, 2001;l(l): 68-76.
22. Michelle T Paulsenl, Adrienne M Starksl, Frederick A Derheimerl,et al. The p53-targeting human phosphatase hCdcl4A interacts with the Cdkl/cyclin B complex and is differentially expressed in human cancers [J]. Mol Cancer, 2006;5(l):25-29.
1.Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J].NATURE,2001;11(6836):494-498.
2.Mailand N,Lukas C,Kaiser BK,et al.Deregulated human Cdcl4A phosphatase disrupts centrosome separation and chromosome segregation[J].Nat Cell Biol,2002;4(4):317-322.
3.Tsafi P,Michael BM,David B.RNA interference[J].Methods,2003,30(4):287-288.
4.Jinek M,Doudna JA.A three-dimensional view of the molecular machinery of RNA interference[J].NATURE,2009,457(7228):405-412.
5.Siomi H,Siomi MC.On the road to reading the RNA-interference code[J].NATURE,2009,457(7228):396-404.
6.Hammond SM,Bemstein E,Beach D,et al.An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells[J].NATURE,2000,404(6775):293-296.
7.Lisa JS,John JR.Approaches for the sequence-specific knockdown of mRNA[J].Nature Biotechnology,2003,21(12):1457-1465
8.Gil J,Esteban M.Induction of apoptosis by the dsRNA-dependent protein kinase(PKR):Mechanism of action[J].Apoptosis,2000,5(2):107-114.
9.Ramaswamy G,Slack FJ.siRNA:A guide for RNA silencing[J].CHEMISTRY & BIOLOGY,2002,9(10):1053-1055.
10.Lipardi C,Wei Q,Paterson BM.RNAi as random degradative PCR:siRNA primers conver mRNA into dsRNAs that are degrate new siRNAs[J].Cell,2001,107(3):297-307.
11.Brett KK,Zachary AZ,Harry C,et al.Disruption of centrosome structure,chromosome segregation,and cytokinesis by misexpression of human Cdc14A phosphatase[J].Molecular Biology of the Ce11,2002;13(7):2289-2300.
12.Mailand N,Lukas C,Kaiser BK,et al.Deregulated human Cdcl4A phosphatase disrupts centrosome separation and chromosome segregation[J].Nat Cell Biol,2002;4(4):317-322.
13.Wu A,Chen J,Baserga R.Nuclear insulin receptor substrate-1 activates promoters of cell cycle progression genes[J].Oncogene,2008,27(3):397-403.
14.Susanne Vetterkinda,Susanne Illenbergerb,Jan Kubicekc,et al.Binding of Par-4 to the actin cytoskeleton is essential for Par-4/Dlk-mediated apoptosis[J].Exp Cell Res,2005,305(2):392-408.
1.Salvatore P,Daniela S,Luisa S,etal.Direct apoptotic effects of free fatty acids on human endothelial cells[J].Nutrition,Metabolism & Cardiovascular Diseases,2008;18(2):96-104.
2.Hermann C,Zeiher AM,Dimmeler S.Shear stress inhibits H~2O~2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase[J].Artherioscler Thromb Vasc Biol,1997;17(12):3588-3592.
3.Ido Y,Carling D,Ruderman N.Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells:inhibition by the AMP-activated protein kinase activation[J].Diabetes,2002;51(1):159-167.
4.Dimmeler S,Haendeler J,Galle J.Oxidized low-density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-1ike proteases:a mechanistic clue to the response to injury hypothesis[J].Circulation,1997;95(2):1760-1763.
5.Shigenaga MK,Hagen TM,Ames BN.Oxidative damage and mitochondrial decay in aging[J].Proc Natl Acad Sci USA,1994;91(23):10771-10778.
6.Boveris A,Valdez LB,Zaobornyj T,et al.Mitochondrialmetabolic states regulate nitric oxide and hydrogen peroxide diffusion to the cytosol[J].Biochim Biophys Acta,2006;1757(5):535-542.
7.Rosen P,Nawroth PP,King G,et al.The role of oxidative stress in the onset and progression of diabetes and its complications:a summary of a Congress Series sponsored by UNESCO-MCBN,the American Diabetes Association and the German Diabetes Society[J].Diabetes Metab Res Rev,2001;17(3):189-212.
8.Chan PH.Reactive oxygen radicals in signaling and damage in the ischemic brain[J].J Cereb Blood Flow Metab,2001; 21(1):2-14. .
9. Lievre V, Becuwe P, Bianchi A, et al. Free radical production and changes in superoxide dismutases associated with hypoxia/reoxygenation-induced apoptosis of embryonic rat forebrain neurons in culture[J]. Free Radic Biol Med,2000; 29(12): 1291-1301.
10. Chan PH. Reactive oxygen radicals in signaling and damage in the Ischemic brain[J]. Cereb Blood Flow Metab, 2001; 21(1): 2-14.
11. Sugawara T, Chan PH. Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia[J].Antioxid Redox Signal, 2003; 5(5): 597-607.
12. Christophe M, Nicolas S. Mitochondria: a target for neuroprotective interventions in cerebral ischemia - reperfusion[J]. Curr Pharm Des,2006; 12(6): 739-757.
13. Sauer H, Wartenberg M. Reactive oxygen species as signaling molecules in cardiovascular differentiation of embryonic stem cells and tumor-induced angiogenesis[J]. Antioxid Redox Signal, 2005; 7(11): 1423-1434.
14. Engel RH, Evens AM. Oxidative stress and apoptosis: a new treatment paradigm in cancer[J]. Front Biosci, 2006; 11(1): 300-312.
15. Bernardi P. Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore by the proton electrochemical gradient. Evidence that the pore can be opened by membrane depolarization[J]. J Biol Chem, 1992; 267(13):8834-8839.
16. Maciel EN, Vercesi AE, Castilho RF. Oxidative stress in Ca(2+)-induced membrane permeability transition in brain mitochondria[J]. J Neurochem, 2001; 79(6):1237-1245.
17. Crompton M. The mitochondrial permeability transition pore and its role in cell death[J]. Biochem J, 1999; 341(2):233-249.
18. Kowaltowski AJ, Castilho RF, Vercesi AE. Mitochondrial permeability transition and oxidative stress[J]. FEBS letters, 2001; 495(1):12-15.
19. Vercesi AE,Kowaltowski AJ, Oliveira HC, et al. Mitochondrial Ca2+ transport, permeability transition and oxidative stress in cell death: implications in cardiotoxicity, neurodegeneration and dyslipidemias [J].Front Biosci, 2006; 11(12):2554-2564.
20. Iadecola C. Bright and dark sides of nitric oxide in ischemic brain damage[J].Trends Neurosci, 1997; 20 (3): 132-139.
21. Christophe M, Nicolas S. Mitochondria: a target for neuroprotective interventions in cerebral ischemia-reperfusion[J]. Curr Pharm Des, 2006; 12 (6): 739-757.
22. Dawson TM, Dawson VL, Synder SH. A novel neuronal messenger molecule in brain: the free radical, nitric oxide[J]. Ann Neurol, 1992; 32(3): 297-311.
23. GUZIK TJ, KORBUT R, ADAMEK GT.Nitric oxide and superoxide in inflammation and immune regulation [J] . J Physiol Pharmacol, 2003; 54 (4) :469-487.
24. Huang Z, Huang PL, Ma J, et al. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine[J]. J Cereb Blood Flow Metab, 1996; 16(5): 981-987.
25. ZengG, Nystrom FH, Ravichandran LV, et al. Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells[J]. Circulation, 2000; 101(13): 1539-1545.
26. Dimmeler S, Fleming I, Fisslthaler B, et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation[J]. Nature, 1999; 399(6736):601-605.
27. Schabitz WR, Kollmar R, Schwaninger M, et al. Neurop rotective effect of granulocyte colony stimulating factor after focal cerebral ischemia[J]. Stroke, 2003; 34(3):745-751.
28. Park HK, Chu K, Lee ST. Granulocyte colony-stimulating factor induces sensorimotor recovery in intracerebral hemorrhage[J]. Brain Res, 2005; 1041 (2): 125-131.
29. Bussolino F, Wang JM, Defilippi P, et al. Granulocyte and granulocyte macrophage colony stimulating factors induce human endothelial cells to migrate and prolixferate[J].Nature, 1989; 337(2):471-473.
30. Krupinski J, Kaluza J, Kumar P, et al. Role of angiogenesis in patients with cerebral ischemic stroke[J]. Stroke, 1994; 25(9):1794-1798.
31. Hartung T. Anti-inflammatory effects of granulocyte colony stimulating factor[J].Curr Op in Hematol,1998; 5(3):221-225.
32. Li Y, Chen J, Chen XG, et al. Human marrow stromal cell therapy for stroke in rat: Neurotrophins and functional recovery[J]. Neurology, 2002; 59(4): 514-523.
33. Schneider A, Kruger C, Steigleder T, et al. The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis[J]. J Clin Invest,2005; 115(8):2083-2098.
34. Ren JM, Fink lestein SP. Growth factor treatment of stroke[J]. Curr Drug Targets CNS Neuro Disord,2005; 4(2):121-125.
35. Maddika S, Ande SR, Panigrahi S,et al. Cell survival, cell death and cell cycle pathways are interconnected: implications for cancer therapy[J]. Drug Resist Updat, 2007; 10(1):13-29.
36. Cantley LC. The phosphoinositide 3-kinase pathway[J]. Science (New York, N. Y 2002; 296(5573):1655-1657.
37. Sussman M. "AKT"ing lessons for stem cells: regulation of cardiac myocyte and progenitor cell proliferation[J]. Trends Cardiovasc Med, 2007; 17(7):235-240.
38. Tassigny A, Berdeaux A, Souktani R, et al. The volume-sensitive chloride channel inhibitors prevent both contractile dysfunction and apoptosis induced by doxorubicin through PI3kinase, Akt and Erk l/2[J].Eur J Heart Fail, 2008; 10(1):39-46.
39. Liu XB, Jiang J, Gui C, et al. Angiopoietin-1 protects mesenchymal stem cells against serum deprivation and hypoxia-induced apoptosis through the PI3K/Akt pathway[J]. Acta pharmacologica Sinica, 2008; 29(7): 815-822.
40. Martelli AM, Faenza I, Billi AM, et al. Intranuclear 3' -phosphoinositide metabolism and Akt signaling: new mechanisms for tumorigenesis and protection against apoptosis[J]? Cellular signallin , 2006 ; 18(8): 1101-1107.
41. Asanuma H, Torigoe T, Kamiguchi K, et al. Surviving expression is regulated by coexpression of human epidermal growth factor receptor 2 and epidermal growth factor receptor via phosphatidylinositol 3-kinase/AKT signaling pathway in breast cancer cells[J]. Cancer Res, 2005; 65(23):11018-11025.
42. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery[J]. Cell, 1997; 91(3):231-241.
43. Hermann C, Assmus B, Urbich C, et al. Insulin-mediated stimulation of protein kinase Akt: A potent survival signaling cascade for endothelial cells[J]. Arterioscler Thromb Vasc Biol, 2000; 20:402-409.
44. Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal kinase (JNK)—from inflammation to development[J]. Curr Opin Cell Biol, 1998; 10(2): 205-219.
45. Xia Z, Dickens M, Raingeaud J, et al. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis[J]. Science, 1995; 270(5240): 1326-1331.
46.Eguchi S.Intracellular signaling of angiotensin Ⅱ-induced p70 S6 kinase phosphorylation at Ser (411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal regulated kinase, and Akt[J].J Biol Chem, 1999, 274(52):8432-8451.
47. Hansen CA, Bartek J, Jensen S. A functional link between the human cell cycle-regulatory phosphatase Cdcl4A and the atypical mitogen-activated kinase Erk3[J].Cell Cycle, 2008, 7(3):325-334.
48. Coulombe P, Rodier G, Pelletier S, et al. Rapid turnover of extracellular signal-regulated kinase 3 by the ubiquitin-proteasome pathway defines a novel paradigm of mitogen-activated protein kinase regulation during cellular difFerentiation[J]. Mol Cell Biol, 2003; 23(13): 4542-4558.
49.Boulton TG, Nye SH, Robbins DJ,et al.ERKs: a family of protein- serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF[J]. Cell, 1991 ; 65(4): 663-675.
50. Crowe DL. Induction of p97MAPK expression regulates collagen mediated inhibition of proliferation and migration in human squamous cell carcinoma lines[J].Int J Oncol, 2004; 24(5):1159-1163.
51. Sun M, Wei Y, Yao L, et al. Identification of extracellular signal-regulated kinase 3 as a new interaction partner of cyclin D3 [J]. Biochem Biophys Res Commun, 2006; 340(1): 209-214.
52. Kummer JL, Rao PK, Heidenreich KA. Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase[J].J Biol Chem, 1997; 272(33): 20490-20494.
53. Herdegen T, Claret FX, Kallunki T, et al. Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury[J]. J Neurosci 1998; 18(14): 5124-5135.
54. Irving EA, Barone FC, Reith AD, et al. Differential activation of MAPK/ERK and p38/SAPK in neurons and glia following focal cerebral ischemia in the rat[J].Mol Brain Res 2000; 77(1):65-75.
55. Barone FC, Irving EA, Ray AM, et al. SB 239063:a second-generation p38 Mitogen-Activated Protein Kinase inhibitor, reduces brain injury and neurological deficits in cerebral focal ischemia[J]. J Pharmacol Exp.Ther, 2001; 296(2) :312-321.
56. Sugino T, Nozaki K,Takagi Y, et al. Activation of mitogenactivated protein kinases after transient forebrain ischemia in gerbil hippocampus[J]. Neurosci, 2001; 20(12):4506-4514.
57. Michael B, Tricia F, Tahir N, et al. Normal neutrophil maturation is associated with selective loss of MAP kinase activation by G-CSF[J]. Leukemia research, 2005; 29(1): 73-78.
1 . Renard C, Van Obberghen E.Role of diabetes in atherosclerotic pathogenesis,What have we learned from animal models[J]? Diabetes Metab,2006; 32(1): 15-29.
2. Kissela BM, Khoury J, Kleindorfer D, et al.Epidemiology of ischaemic stroke in patients with diabetes:the greater Cincinnati/northern Kentucky Stroke Study[J]. Diabetes Care, 2005; 28(2):355-359.
3. Baird TA, Parsons MW, Barber PA, et al. The influence of diabetes mellitus and hyperglycaemia on stroke incidence and outcome[J].J Clin Neurosci, 2002; 9(6):618-626.
4. Giorda CB, Avogaro A, Maggini M, et al. Incidence and risk factors for stroke in type 2 diabetic patients:The DAI Study[J]. Stroke, 2007; 38(4): 1154-1160.
5. Malmberg K, Yusuf S, Gerstein HC, et al. Impact of diabetes on long-term prognosis in patients with unstable angina and non-Q-wave myocardial infarction:Results of the OASIS (Organization to A ssess Strategies for Ischemic Syndromes) Registry[J]. Circulation, 2000 ; 102(9):1014-1019.
6. Koren-Morag N, Goldbourt U, Tanne D. Relationship between the metabolic syndrome and ischemic stroke or transient ischemic attack[J]. Stroke, 2005; 36(7): 1366-1371.
7. DECODE Study Group. Gender difference in all-cause and cardiovascular mortality related to hyperglycaemia and newly-diagnosed diabetes[J]. Diabetologia,2003; 46(5):608-617.
8. Tuomilehto J, RastenytD, Jousilahti P, et al. Diabetes mellitus as a risk factor for death from stroke prospective study of the middle-aged Finnish population[J]. Stroke, 1996; 27(2): 210-215.
9. Almdal T, Scharling H, Jensen JS, et al. The independent effect of type 2 diabetes mellitus on ischemic heart disease, stroke, and death.A population-based study of 13000 men and women with 20 years of follow-up[J]. Arch Intern Med, 2004; 164(13):1422-1426.
10. Ho JE, Paultre F, Mosca L, et al. Is diabetes mellitus a cardiovascular disease risk equivalent for fatal stroke in women? Data from the women's pooling project[J]. Stroke,2003; 34(12):2812-2816.
11. Rothwell PM, Giles MF, Flossmann E, et al.A simple score (ABCD) to identify individuals at high early risk of stroke after a transient ischaemic attack[J].Lancet, 2005; 366(9479):29-36.
12. Ferrero E, Zocchi MR, Magni E, et al. Roles of tumor necrosis factor P55 and P75 receptors in TNF-induced vascular permeability[J]. Am JPhysiol Cell, 2001; 281(4):1173-1179.
13. American Diabetes Association. Management of dyslipidemia in adult with diabetes[J]. Diabetes Care, 2002; 25(suppl l):S74-S75.
14. Baron AD, Quon MJ, Reaven GM, et al. Insulin action and endothelial function: Contemporary Endocrinology; Insulin resistance:The metabolic syndrome X[M]. Totowa:Human Press, 1999:247-266.
15. Goldstein BJ. Insulin resistance as the core defect in type 2 diabetes mellitus[J].Am J Cardiol, 2002; 90(5A):3-10.
16. Marc R, Carlos M, Joan M, et al. Acute Hyperglycemia State Is Associated With Lower tPA-Induced Recanalization Rates in Stroke Patients[J]. Stroke,2005; 36 (1):1705-1707.
17. Andolfi A, Giaccari A, Cilli C, et al. Acute hyperglycemia and acute hyperinsulinemia decrease plasma fibrinolytic activity and increase plasminogen activator inhibitor type 1 in the rat[J].Acta Diabetol, 2001; 38(2):71-76.
18. Agullo-Ortufio MT, Albalade jo, Parro S, et al. Plasmatic homocysteine concentration and its relationship with complications associated to diabetes mellitus[J]. Clinical Chimica Acta, 2002; 326(1):105-112.
19. Zhang XD,Chen YR, Ge L, et al. Features of stroke in Chinese diabetes patients: a hospital-based study[J]. J Int Med Res, 2007; 35(4) :540-546.
20. Ortega-Casarrubios MA, Fuentes B, San Jos(?) B, et al. Influence of previous diagnosis of diabetes mellitus in the stroke severity and in-hospital outcome in acute cerebral infarction[J]. Neurologia, 2007; 22(7): 426-433.
21. Pinto A,Tuttolomondo A,Di Raimondo D, et al.A case control study between diabetic and non-diabetic subjects with ischemic stroke[J]. Int Angiol, 2007; 26(1):26-32.
22. Tuttolomondo A, Pinto A, Salemi G, et al.Diabetic and non-diabetic ubjects with ischemic stroke:Differences, subtype distribution and outcome[J]. Nutrition, Metabolism and Cardiovascular Diseases, 2008; 18(2): 152-157.
23. Plengvidhya N, Leelawatana R, Pratipanawatr T, et al. Thailand diabetes registry project: prevalence and risk factors of stroke in thai diabetic patients[J]. J Med Assoc Thai, 2006; 89(Suppl. 1):S49-S53.
24. Matz K, Keresztes K, Tatschl C, et al. Disorders of glucose metabolism in acute stroke patients. An underrecognised problem[J]. Diabetes Care, 2006; 29(4):792-797.
25. Smajlovic D, Salihovic D, Ibrahimagic 0, et al. Stroke in patients with diabetes mellitus:a hospital based study[J].Med Arh, 2006 ; 60(6 Suppl. 2):S63-S65.
26. Khealani BA, Syed NA, Maken S, et al. Predictors of ischemic versus hemorrhagic strokes in hypertensive patients[J]. J Coll Physicians Surg Pak, 2005; 15(1):22-25.
27. Stollberger C, Exner I, Finsterer J, et al. Stroke in diabetic and non-diabetic patients:course and prognostic value of admission serum glucose[J]. Ann Med, 2005; 37 (5):357-364.
28. Zodpey SP, Tiwari RR.A risk scoring system for prediction of haemorrhagic stroke[J]. Indian J Public Health, 2005; 49(4):218-222.
29. Feldmann E, Broderick JP, Kernan WN, et al.Major risk factors for intracerebral hemorrhage in the young are modifiable[J]. Stroke, 2005; 36(9): 1881-1885.
30. Herzig RI, Vlachova JM, Gabrys MD, et al. Occurrence of diabetes mellitus in spontaneous intracerebral hemorrhage[J]. Acta Diabetol, 2007; 44(4): 201-207.
31. Arboix A, Massons J, Garcia-Eroles L, et al. Diabetes is an independent risk factor for in-hospital mortality from acute spontaneous intracerebral hemorrhage[J]. DIABETES CARE,2006; 23(10):1527-1532 .
32. SaccoRL, Adams R, Albers G, et al. Guidelines for prevention of stroke in patients with ischemic stroke or transient ischemic attack: A statement for healthcare professionals from the American Heart Association/American Stroke Association Council on Stroke: Cosponsored by the Council on Cardiovascular Radiology and Intervention: the American Academy of Neurology affirms the value of this guideline[J]. Stroke, 2006; 37(2):577-617.
33. Dalen JE. Aspirin to prevent heart attack and stroke: What' s the right dose?[J]. Am J Med, 2006; 119(3):198-202.
34. He J, Whelton PK, Vu B, et al. Aspirin and risk of hemorrhagic stroke: Ameta-analysis of randomized controlled trials[J]. JAMA, 1998; 280(22): 1930-1935.
35. Hallas J, Dall M, Andries A, et al. Use of single and combined antithrombotic therapy and risk of serious upper gastrointestinal bleeding: Population based case-control study[J]. BMJ, 2006; 333(7571): 726-728.
36. CAPRIE Steering Committee. A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE)[J]. Lancet, 1996; 348 (9038) :1329-1339.
37. Halkes PH, Van Gijn J, Kappelle LJ, et al. Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia of arterial origin (ESPRIT):Randomised controlled trial[J].Lancet, 2006; 367(9523): 1665-1673.
38. Likosky DJ, Lee K, Brown DM, et al.Evidence-based medicine: Review of guidelines and trials in the prevention of secondary stroke[J].J HOSPITAL MEDICINE, 2008; 3 (Suppl.4) :S6-S19.
39. Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. UK Prospective Diabetes Study Group[J]. BMJ, 1998; 317(7160):703-713.
40. Amarenco P, Bogousslavsky J, Callahan A, et al. High-dose atorvastatin after stroke or transient ischemic attack[J]. N Engl J Med, 2006; 355(6) : 549-559.
41. Bokura H, Kobayashi S, Yamaguchi S, et al. Clinical characteristics and prognosis in stroke patients with diabetes mellitus:retrospective evaluation using the Japanese Standard Stroke Registry database (JSSRS) [J]. Brain and Nerve, 2006; 58(2):135-139.
42.Hacke W,Albers G,Al-Rawi Y,et al.The Desmoteplase in Acute Ischemic Stroke Trial (DIAS):A phaseⅡMRI-based 9-hour window acute stroke thrombolysis trial with intravenous desmoteplase[J].Stroke,2005;36(1):66-73.
2.Nasmyth K.Viewpoint:putting the cell cycle in order[J].Science,1996;274(5293):1640-1645.
3.Morgan DO.Cyclin-dependent kinases:engines,clocks,and microprocessors[J].Annu Rev Cell Dev Biol,1997;13(11):261-291.
4.Willamson J,Chang K,Frangos M,et al.Hyperglycemic pseudohypoxia and diabetic complications[J].Diabetes,1993;42(6):801-813.
5.Visintm R,Craig K,Hwang ES,et al.The phosphatase Cdcl4 triggers mitotic exit by reversal of Cdk-dependent phosphorylation [J]. Mol Cell, 1998;2(6): 709-71.
6. Wan J, Xu H, Grunstein M.Cdcl4 of saccharomyces cerevisiae. Cloning, sequence analysis and transcription during the cell cycle [J]. J Biol Chem, 1992; 267(16): 11274-11280.
7. Li L, Ernsting BR, Wishart MJ, et al. A family of putative tumor suppressors is structurally and functionally conserved in humans and yeast [J]. J Biol Chem, 1997;272(47): 29403-29406.
8. Hyekyung P Cho, Yie Liu, Maria Gomez, et al. The dual-specificity phosphatase cdcl4B bundles and stabilizes microtubules[J]. Molecular and Cellular Biology, 2005; 25(1): 4541-4551.
9. Grzegorz Nalepa, J. Wade Harper. Visualization of a highly organized intranuclear network of filament in living mammalian cells [J]. Cell Motility and Cytoskeleton, 2004; 59(2): 94-108.
10. Brett KK, Zachary AZ, Harry C,et al. Disruption of centrosome structure, chromosome segregation, and cytokinesis by misexpression of human Cdcl4A phosphatase [J]. Molecular Biology of the Cell, 2002; 13(7): 2289-2300.
11. Niels Mailand, Claudia Lukas, Brett K, et al. Deregulated human Cdcl4A phosphatase disrupts centrosome separation and chromosome segregation[J]. Nature Cell Biology, 2002; 4(4): 317-322.
12. Vazquez-Novelle MD, Esteban V, Bueno A,et al.Functional homology among human and fission yeast cdcl4 phophatases[J]. J Biol Chem, 2005; 280(32): 29144-29150.
1.Cugnet-Anceau C,Bauduceau B.Glycaemic control and cardiovascular morbi-mortality:The contribution of the 2008 studies[J].Ann Endocrinol(Paris),2009;70(1):48-54.
2.Kissela BM,Khoury J,Kleindorfer D,et al.Epidemiology of ischaemic stroke in patients with diabetes:the greater Cincinnati/northern Kentucky Stroke Study[J].Diabetes Care,2005;28(2):355-359.
3.Ahmad FK,He Z,King GL.Molecular targets of diabetic cardiovascular complications[J].Curr Drug Target,2005;6(4):487-494.
4.De Caterina R.Endothelial dysfunctions:common denominatorsin vascular disease[J].Curr Opin Clin Nutr Metab Care.2000;3(6):453-467.
5.Szrnitko PE,Wang CH,Weisel RD,et al.Biomarkers of vascular disease linking inflammation to endothelial activation:Part Ⅱ[J].Circulation,2003;108(1):2041-2058.
6.Verma S,Buchanan MR,Anderson TJ.Endothelial function testing as a biomarker of vascular disease[J].Circulation,2003;108(1):2054-2069.
7.Visintin R,Craig K,Hwang ES,et al.The phosphatase Cdcl4 triggers mitotic exit by reversal of Cdk-dependent phosphorylation[J].Mol Cell,1998;2(6):709-718.
8.MICHIELS C.Endothelial cell functions[J].J Cell Physiol,2003;196 (3):430-443.
9.Nagy Z,VastagM,Skopal J,et al.Human brain microvessel endothelial cell culture as a model system to study vascular factors ofischemic brain[J].Keio-J-Med,1996;45(3):200-206.
10.D'Agnillo F,Wood F,Porras C,et al.Effects ofhypoxia and glutathione depletion on hemoglobin and myoglobin-mediated oxidative stress toward endothelium[J].Bichim Biophys Acta,2000;1495(2):150-159.
11.Samarasinghe DA,Tapner M,Ferrell GC.Role of oxidative stress on hypoxia-reoxygenation injury to cultured rat hepatic sinusoidal endothelial cells[J].Hepatology,2000;31(1):160-165.
12.Shostak HDC,Lemasters JJ,Edgell CJ,et al.Role oflCElike proteases in endothelial cell hypoxic and reperfusion injury[J].BiochemBiophys ResCommun,1997;231(3):844-847.
13.Faller DV.Endothelial cell responses to hypoxic stress[J].Clin Exp Pharmacol Physiol,1999;26(1):74-84.
14.Brett KK,Zachary AZ,Harry C,et al.Disruption of centrosorre structure,chromosome segregation,and cytokinesis by misexpression of human Cdcl4A phosphatase[J].Molecular Biology of the Cell,2002;13(7):2289-2300.
15.Vazquez-Novelle MD,Esteban V,Bueno A,et al.Functional homology among human and fission yeast cdcl4 phophatases[J].J Biol Che,2005;280(32):29144-29150.
16.Fields S,Song O-K A novel genetic system to detect protein-protein interactions[J].Nature,1989;340(6230):245-246.
17.Golemis E.Protein-protein interactions,A Molecular Cloning Manual[M].New York:Cold Spring Harbour Laboratory press, 2002:1-682.
18. Bartel PL, Fields S. The yeast two-hybrid system [M]. New York: Oxford University Press, 1997:425-428.
19. Sandrock B, Tirode F, Egly JM. Methods in Molecular Biology: Two-Hybrid System: Methods and Protocols[M]. New Jersey: Human press, 2001:271-289.
20. Dawai T,Matsumoto M,Takeda K,et al.ZIP kinase a novel serine/threonine kinase which mediates apoptosis [J]. Mol Cell Biol, 1998; 18(3): 1642-1651.
21. Kogel D, Reimertz C, Dussmann H, et al. The death associated protein (DAP) kinase homologue Dlk/ZIP kinase induces pl9ARF-and p53-independent apoptosis[J]. Eur J Cance, 2003; 39(2):249-256.
22. Kogel D, Plottner O, Landsberg G et al. Cloning and characterization of Dlk, a novel serine/threonine kinase that is tightly associated with chromatin and phophorylates core histones [J]. Oncogene, 1998; 17(20): 2645-2654.
1.Wolf G Molecular mechanisms of renal hypertrophy:role of p27Kipl[J].Kidney Int,1999;56(4):1262-1265.
2.Brett KK,Zachary AZ,Harry C,et al.Disruption of Centrosome Structure,Chromosome Segregation,and Cytokinesis by Misexpression of Human Cdc14A Phosphatase[J].Molecular Biology of the Cell,2002;13(7):2289-2300.
3.Piel,M,Nordberg,J,Euteneuer,U,Bomens,M.Centrosome-dependent exit of cytokinesis in animal cells[J].Science,2001;291(5508):1550-1553.
4.Hyekyung P Cho,Yie Liu,Maria Gomez,et al.The dual-specificity phosphatase cdc14B bundles and stabilizes microtubules[J].Molecular and Cellular Biology,2005;25(1):4541-4551.
5.Pockwinse SM,Krockmalnic G,Doxsey S J,Nickerson J,et al.Cell cycle independent interaction of CDC2 with the centrosome,which is associated with the nuclear matrix-intermediate filament scaffold[J].Proc Natl Acad Sci,1997;94(7):3022-3027.
6.Okuda M,Hom HF,Tarapore P,et al.Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication[J].Cell,2000;103(1):127-140.
7.Michelle T P,Adrienne M S,Frederick A D,et al.The p53-targeting human phosphatase hCdcl4A interacts with the Cdkl/cyclin B complex and is diffententially expressed in human cancers[J].Molecular Cancer,2006;5(25):2100-2110.
8.Brett K.Kaiser,Zachary A.Zimmerman,Harry Charbonneau,et al.Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdcl4A Phosphatase[J]. Mol Biol Cell, 2002;13(7): 2289-2300.
9. Bembenek J, Yu H. Regulation of the anaphase-promoting complex by the dual specificity phosphatase human Cdcl4a[J].J. Biol. Chem, 2001;276(51): 48237-48242.
10. Brett K. Kaiser, Zachary A. Zimmerman, Harry Charbonneau, et al. Disruption of centrosome structure, chromosome segregation, and cytokinesis by misexpression of human Cdcl4A phosphatase[J]. Molecular Biology of the Cell, 2002;13(7): 2289-2300.
11. Mailand N, Lukas C, Kaiser BK, Jackson PK, Bartek J, Lukas J. Deregulated human Cdcl4A phosphatase disrupts centrosome separation and chromosome segregation [J]. Nat. Cell Biol, 2002;4(4):317-322.
12. M. Dolores Va' zquez-Novelle, Vero' nica Esteban, Avelino Bueno, Man'a P. Sacristan. Functional Homology among Human and Fission Yeast Cdcl4 Phosphatases [J]. J Biol Chem, 2005;280(32): 29144-29150.
13. Martinsson HS ,Starborg M, Erlandsson F. et al. Single cell analysis of Gl check points-the relationship between the restriction point and phosphorylation of pRb [J]. Exp Cell Res, 2005;305(2):383-391.
14. Keyomarsi K, Herliczek TW. The role of Cyclin E in cell proliferation, development and cancer [J]. Prog Cell Cycle Res,1997;3(l): 171-191.
15. Montero S, Guzman C,Vargas C, et al. Prognostic value of cytosolic p53 protein in breast cancer [J]. Tumor Biol, 2001;22 (5): 337-344.
16. Burns TF, El-deiry WS. The P53 pathway and apoptosis[J].J Cell Physiol, 1999;181(2): 231-239.
17. Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage [J]. Science, 1998;282(5393): 1497-1501.
18. Barboule N, Chadebech P, Baldin V, et al. Involvement of p21 in mitotic exit after paclitaxel treatment in MCF-7 breast adenocarcinoma cell line[J].Oncogene, 1997;15(23): 2867-2875.
19. Li L, Ljungman M, Dixon JE. The human Cdcl4 phosphatases interact with and dephosphorylate the tumor suppressor protein p53 [J]. J. Biol. Chem, 2000;275(4): 2410-2414.
20. Lowe SW, Bodis S, Bardeesy N, et al. Apoptosis and the prognostic significance of P53 mutation [J]. Cold Spring Harb Symp Quant Biol, 1994;59: 419-426.
21. Bullock AN, Fersht AR. Rescuing the function of mutant p53 [J]. Nat Rev Cancer, 2001;l(l): 68-76.
22. Michelle T Paulsenl, Adrienne M Starksl, Frederick A Derheimerl,et al. The p53-targeting human phosphatase hCdcl4A interacts with the Cdkl/cyclin B complex and is differentially expressed in human cancers [J]. Mol Cancer, 2006;5(l):25-29.
1.Elbashir SM,Harborth J,Lendeckel W,et al.Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[J].NATURE,2001;11(6836):494-498.
2.Mailand N,Lukas C,Kaiser BK,et al.Deregulated human Cdcl4A phosphatase disrupts centrosome separation and chromosome segregation[J].Nat Cell Biol,2002;4(4):317-322.
3.Tsafi P,Michael BM,David B.RNA interference[J].Methods,2003,30(4):287-288.
4.Jinek M,Doudna JA.A three-dimensional view of the molecular machinery of RNA interference[J].NATURE,2009,457(7228):405-412.
5.Siomi H,Siomi MC.On the road to reading the RNA-interference code[J].NATURE,2009,457(7228):396-404.
6.Hammond SM,Bemstein E,Beach D,et al.An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells[J].NATURE,2000,404(6775):293-296.
7.Lisa JS,John JR.Approaches for the sequence-specific knockdown of mRNA[J].Nature Biotechnology,2003,21(12):1457-1465
8.Gil J,Esteban M.Induction of apoptosis by the dsRNA-dependent protein kinase(PKR):Mechanism of action[J].Apoptosis,2000,5(2):107-114.
9.Ramaswamy G,Slack FJ.siRNA:A guide for RNA silencing[J].CHEMISTRY & BIOLOGY,2002,9(10):1053-1055.
10.Lipardi C,Wei Q,Paterson BM.RNAi as random degradative PCR:siRNA primers conver mRNA into dsRNAs that are degrate new siRNAs[J].Cell,2001,107(3):297-307.
11.Brett KK,Zachary AZ,Harry C,et al.Disruption of centrosome structure,chromosome segregation,and cytokinesis by misexpression of human Cdc14A phosphatase[J].Molecular Biology of the Ce11,2002;13(7):2289-2300.
12.Mailand N,Lukas C,Kaiser BK,et al.Deregulated human Cdcl4A phosphatase disrupts centrosome separation and chromosome segregation[J].Nat Cell Biol,2002;4(4):317-322.
13.Wu A,Chen J,Baserga R.Nuclear insulin receptor substrate-1 activates promoters of cell cycle progression genes[J].Oncogene,2008,27(3):397-403.
14.Susanne Vetterkinda,Susanne Illenbergerb,Jan Kubicekc,et al.Binding of Par-4 to the actin cytoskeleton is essential for Par-4/Dlk-mediated apoptosis[J].Exp Cell Res,2005,305(2):392-408.
1.Salvatore P,Daniela S,Luisa S,etal.Direct apoptotic effects of free fatty acids on human endothelial cells[J].Nutrition,Metabolism & Cardiovascular Diseases,2008;18(2):96-104.
2.Hermann C,Zeiher AM,Dimmeler S.Shear stress inhibits H~2O~2-induced apoptosis of human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase[J].Artherioscler Thromb Vasc Biol,1997;17(12):3588-3592.
3.Ido Y,Carling D,Ruderman N.Hyperglycemia-induced apoptosis in human umbilical vein endothelial cells:inhibition by the AMP-activated protein kinase activation[J].Diabetes,2002;51(1):159-167.
4.Dimmeler S,Haendeler J,Galle J.Oxidized low-density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-1ike proteases:a mechanistic clue to the response to injury hypothesis[J].Circulation,1997;95(2):1760-1763.
5.Shigenaga MK,Hagen TM,Ames BN.Oxidative damage and mitochondrial decay in aging[J].Proc Natl Acad Sci USA,1994;91(23):10771-10778.
6.Boveris A,Valdez LB,Zaobornyj T,et al.Mitochondrialmetabolic states regulate nitric oxide and hydrogen peroxide diffusion to the cytosol[J].Biochim Biophys Acta,2006;1757(5):535-542.
7.Rosen P,Nawroth PP,King G,et al.The role of oxidative stress in the onset and progression of diabetes and its complications:a summary of a Congress Series sponsored by UNESCO-MCBN,the American Diabetes Association and the German Diabetes Society[J].Diabetes Metab Res Rev,2001;17(3):189-212.
8.Chan PH.Reactive oxygen radicals in signaling and damage in the ischemic brain[J].J Cereb Blood Flow Metab,2001; 21(1):2-14. .
9. Lievre V, Becuwe P, Bianchi A, et al. Free radical production and changes in superoxide dismutases associated with hypoxia/reoxygenation-induced apoptosis of embryonic rat forebrain neurons in culture[J]. Free Radic Biol Med,2000; 29(12): 1291-1301.
10. Chan PH. Reactive oxygen radicals in signaling and damage in the Ischemic brain[J]. Cereb Blood Flow Metab, 2001; 21(1): 2-14.
11. Sugawara T, Chan PH. Reactive oxygen radicals and pathogenesis of neuronal death after cerebral ischemia[J].Antioxid Redox Signal, 2003; 5(5): 597-607.
12. Christophe M, Nicolas S. Mitochondria: a target for neuroprotective interventions in cerebral ischemia - reperfusion[J]. Curr Pharm Des,2006; 12(6): 739-757.
13. Sauer H, Wartenberg M. Reactive oxygen species as signaling molecules in cardiovascular differentiation of embryonic stem cells and tumor-induced angiogenesis[J]. Antioxid Redox Signal, 2005; 7(11): 1423-1434.
14. Engel RH, Evens AM. Oxidative stress and apoptosis: a new treatment paradigm in cancer[J]. Front Biosci, 2006; 11(1): 300-312.
15. Bernardi P. Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore by the proton electrochemical gradient. Evidence that the pore can be opened by membrane depolarization[J]. J Biol Chem, 1992; 267(13):8834-8839.
16. Maciel EN, Vercesi AE, Castilho RF. Oxidative stress in Ca(2+)-induced membrane permeability transition in brain mitochondria[J]. J Neurochem, 2001; 79(6):1237-1245.
17. Crompton M. The mitochondrial permeability transition pore and its role in cell death[J]. Biochem J, 1999; 341(2):233-249.
18. Kowaltowski AJ, Castilho RF, Vercesi AE. Mitochondrial permeability transition and oxidative stress[J]. FEBS letters, 2001; 495(1):12-15.
19. Vercesi AE,Kowaltowski AJ, Oliveira HC, et al. Mitochondrial Ca2+ transport, permeability transition and oxidative stress in cell death: implications in cardiotoxicity, neurodegeneration and dyslipidemias [J].Front Biosci, 2006; 11(12):2554-2564.
20. Iadecola C. Bright and dark sides of nitric oxide in ischemic brain damage[J].Trends Neurosci, 1997; 20 (3): 132-139.
21. Christophe M, Nicolas S. Mitochondria: a target for neuroprotective interventions in cerebral ischemia-reperfusion[J]. Curr Pharm Des, 2006; 12 (6): 739-757.
22. Dawson TM, Dawson VL, Synder SH. A novel neuronal messenger molecule in brain: the free radical, nitric oxide[J]. Ann Neurol, 1992; 32(3): 297-311.
23. GUZIK TJ, KORBUT R, ADAMEK GT.Nitric oxide and superoxide in inflammation and immune regulation [J] . J Physiol Pharmacol, 2003; 54 (4) :469-487.
24. Huang Z, Huang PL, Ma J, et al. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine[J]. J Cereb Blood Flow Metab, 1996; 16(5): 981-987.
25. ZengG, Nystrom FH, Ravichandran LV, et al. Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells[J]. Circulation, 2000; 101(13): 1539-1545.
26. Dimmeler S, Fleming I, Fisslthaler B, et al. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation[J]. Nature, 1999; 399(6736):601-605.
27. Schabitz WR, Kollmar R, Schwaninger M, et al. Neurop rotective effect of granulocyte colony stimulating factor after focal cerebral ischemia[J]. Stroke, 2003; 34(3):745-751.
28. Park HK, Chu K, Lee ST. Granulocyte colony-stimulating factor induces sensorimotor recovery in intracerebral hemorrhage[J]. Brain Res, 2005; 1041 (2): 125-131.
29. Bussolino F, Wang JM, Defilippi P, et al. Granulocyte and granulocyte macrophage colony stimulating factors induce human endothelial cells to migrate and prolixferate[J].Nature, 1989; 337(2):471-473.
30. Krupinski J, Kaluza J, Kumar P, et al. Role of angiogenesis in patients with cerebral ischemic stroke[J]. Stroke, 1994; 25(9):1794-1798.
31. Hartung T. Anti-inflammatory effects of granulocyte colony stimulating factor[J].Curr Op in Hematol,1998; 5(3):221-225.
32. Li Y, Chen J, Chen XG, et al. Human marrow stromal cell therapy for stroke in rat: Neurotrophins and functional recovery[J]. Neurology, 2002; 59(4): 514-523.
33. Schneider A, Kruger C, Steigleder T, et al. The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis[J]. J Clin Invest,2005; 115(8):2083-2098.
34. Ren JM, Fink lestein SP. Growth factor treatment of stroke[J]. Curr Drug Targets CNS Neuro Disord,2005; 4(2):121-125.
35. Maddika S, Ande SR, Panigrahi S,et al. Cell survival, cell death and cell cycle pathways are interconnected: implications for cancer therapy[J]. Drug Resist Updat, 2007; 10(1):13-29.
36. Cantley LC. The phosphoinositide 3-kinase pathway[J]. Science (New York, N. Y 2002; 296(5573):1655-1657.
37. Sussman M. "AKT"ing lessons for stem cells: regulation of cardiac myocyte and progenitor cell proliferation[J]. Trends Cardiovasc Med, 2007; 17(7):235-240.
38. Tassigny A, Berdeaux A, Souktani R, et al. The volume-sensitive chloride channel inhibitors prevent both contractile dysfunction and apoptosis induced by doxorubicin through PI3kinase, Akt and Erk l/2[J].Eur J Heart Fail, 2008; 10(1):39-46.
39. Liu XB, Jiang J, Gui C, et al. Angiopoietin-1 protects mesenchymal stem cells against serum deprivation and hypoxia-induced apoptosis through the PI3K/Akt pathway[J]. Acta pharmacologica Sinica, 2008; 29(7): 815-822.
40. Martelli AM, Faenza I, Billi AM, et al. Intranuclear 3' -phosphoinositide metabolism and Akt signaling: new mechanisms for tumorigenesis and protection against apoptosis[J]? Cellular signallin , 2006 ; 18(8): 1101-1107.
41. Asanuma H, Torigoe T, Kamiguchi K, et al. Surviving expression is regulated by coexpression of human epidermal growth factor receptor 2 and epidermal growth factor receptor via phosphatidylinositol 3-kinase/AKT signaling pathway in breast cancer cells[J]. Cancer Res, 2005; 65(23):11018-11025.
42. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery[J]. Cell, 1997; 91(3):231-241.
43. Hermann C, Assmus B, Urbich C, et al. Insulin-mediated stimulation of protein kinase Akt: A potent survival signaling cascade for endothelial cells[J]. Arterioscler Thromb Vasc Biol, 2000; 20:402-409.
44. Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal kinase (JNK)—from inflammation to development[J]. Curr Opin Cell Biol, 1998; 10(2): 205-219.
45. Xia Z, Dickens M, Raingeaud J, et al. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis[J]. Science, 1995; 270(5240): 1326-1331.
46.Eguchi S.Intracellular signaling of angiotensin Ⅱ-induced p70 S6 kinase phosphorylation at Ser (411) in vascular smooth muscle cells. Possible requirement of epidermal growth factor receptor, Ras, extracellular signal regulated kinase, and Akt[J].J Biol Chem, 1999, 274(52):8432-8451.
47. Hansen CA, Bartek J, Jensen S. A functional link between the human cell cycle-regulatory phosphatase Cdcl4A and the atypical mitogen-activated kinase Erk3[J].Cell Cycle, 2008, 7(3):325-334.
48. Coulombe P, Rodier G, Pelletier S, et al. Rapid turnover of extracellular signal-regulated kinase 3 by the ubiquitin-proteasome pathway defines a novel paradigm of mitogen-activated protein kinase regulation during cellular difFerentiation[J]. Mol Cell Biol, 2003; 23(13): 4542-4558.
49.Boulton TG, Nye SH, Robbins DJ,et al.ERKs: a family of protein- serine/threonine kinases that are activated and tyrosine phosphorylated in response to insulin and NGF[J]. Cell, 1991 ; 65(4): 663-675.
50. Crowe DL. Induction of p97MAPK expression regulates collagen mediated inhibition of proliferation and migration in human squamous cell carcinoma lines[J].Int J Oncol, 2004; 24(5):1159-1163.
51. Sun M, Wei Y, Yao L, et al. Identification of extracellular signal-regulated kinase 3 as a new interaction partner of cyclin D3 [J]. Biochem Biophys Res Commun, 2006; 340(1): 209-214.
52. Kummer JL, Rao PK, Heidenreich KA. Apoptosis induced by withdrawal of trophic factors is mediated by p38 mitogen-activated protein kinase[J].J Biol Chem, 1997; 272(33): 20490-20494.
53. Herdegen T, Claret FX, Kallunki T, et al. Lasting N-terminal phosphorylation of c-Jun and activation of c-Jun N-terminal kinases after neuronal injury[J]. J Neurosci 1998; 18(14): 5124-5135.
54. Irving EA, Barone FC, Reith AD, et al. Differential activation of MAPK/ERK and p38/SAPK in neurons and glia following focal cerebral ischemia in the rat[J].Mol Brain Res 2000; 77(1):65-75.
55. Barone FC, Irving EA, Ray AM, et al. SB 239063:a second-generation p38 Mitogen-Activated Protein Kinase inhibitor, reduces brain injury and neurological deficits in cerebral focal ischemia[J]. J Pharmacol Exp.Ther, 2001; 296(2) :312-321.
56. Sugino T, Nozaki K,Takagi Y, et al. Activation of mitogenactivated protein kinases after transient forebrain ischemia in gerbil hippocampus[J]. Neurosci, 2001; 20(12):4506-4514.
57. Michael B, Tricia F, Tahir N, et al. Normal neutrophil maturation is associated with selective loss of MAP kinase activation by G-CSF[J]. Leukemia research, 2005; 29(1): 73-78.
1 . Renard C, Van Obberghen E.Role of diabetes in atherosclerotic pathogenesis,What have we learned from animal models[J]? Diabetes Metab,2006; 32(1): 15-29.
2. Kissela BM, Khoury J, Kleindorfer D, et al.Epidemiology of ischaemic stroke in patients with diabetes:the greater Cincinnati/northern Kentucky Stroke Study[J]. Diabetes Care, 2005; 28(2):355-359.
3. Baird TA, Parsons MW, Barber PA, et al. The influence of diabetes mellitus and hyperglycaemia on stroke incidence and outcome[J].J Clin Neurosci, 2002; 9(6):618-626.
4. Giorda CB, Avogaro A, Maggini M, et al. Incidence and risk factors for stroke in type 2 diabetic patients:The DAI Study[J]. Stroke, 2007; 38(4): 1154-1160.
5. Malmberg K, Yusuf S, Gerstein HC, et al. Impact of diabetes on long-term prognosis in patients with unstable angina and non-Q-wave myocardial infarction:Results of the OASIS (Organization to A ssess Strategies for Ischemic Syndromes) Registry[J]. Circulation, 2000 ; 102(9):1014-1019.
6. Koren-Morag N, Goldbourt U, Tanne D. Relationship between the metabolic syndrome and ischemic stroke or transient ischemic attack[J]. Stroke, 2005; 36(7): 1366-1371.
7. DECODE Study Group. Gender difference in all-cause and cardiovascular mortality related to hyperglycaemia and newly-diagnosed diabetes[J]. Diabetologia,2003; 46(5):608-617.
8. Tuomilehto J, RastenytD, Jousilahti P, et al. Diabetes mellitus as a risk factor for death from stroke prospective study of the middle-aged Finnish population[J]. Stroke, 1996; 27(2): 210-215.
9. Almdal T, Scharling H, Jensen JS, et al. The independent effect of type 2 diabetes mellitus on ischemic heart disease, stroke, and death.A population-based study of 13000 men and women with 20 years of follow-up[J]. Arch Intern Med, 2004; 164(13):1422-1426.
10. Ho JE, Paultre F, Mosca L, et al. Is diabetes mellitus a cardiovascular disease risk equivalent for fatal stroke in women? Data from the women's pooling project[J]. Stroke,2003; 34(12):2812-2816.
11. Rothwell PM, Giles MF, Flossmann E, et al.A simple score (ABCD) to identify individuals at high early risk of stroke after a transient ischaemic attack[J].Lancet, 2005; 366(9479):29-36.
12. Ferrero E, Zocchi MR, Magni E, et al. Roles of tumor necrosis factor P55 and P75 receptors in TNF-induced vascular permeability[J]. Am JPhysiol Cell, 2001; 281(4):1173-1179.
13. American Diabetes Association. Management of dyslipidemia in adult with diabetes[J]. Diabetes Care, 2002; 25(suppl l):S74-S75.
14. Baron AD, Quon MJ, Reaven GM, et al. Insulin action and endothelial function: Contemporary Endocrinology; Insulin resistance:The metabolic syndrome X[M]. Totowa:Human Press, 1999:247-266.
15. Goldstein BJ. Insulin resistance as the core defect in type 2 diabetes mellitus[J].Am J Cardiol, 2002; 90(5A):3-10.
16. Marc R, Carlos M, Joan M, et al. Acute Hyperglycemia State Is Associated With Lower tPA-Induced Recanalization Rates in Stroke Patients[J]. Stroke,2005; 36 (1):1705-1707.
17. Andolfi A, Giaccari A, Cilli C, et al. Acute hyperglycemia and acute hyperinsulinemia decrease plasma fibrinolytic activity and increase plasminogen activator inhibitor type 1 in the rat[J].Acta Diabetol, 2001; 38(2):71-76.
18. Agullo-Ortufio MT, Albalade jo, Parro S, et al. Plasmatic homocysteine concentration and its relationship with complications associated to diabetes mellitus[J]. Clinical Chimica Acta, 2002; 326(1):105-112.
19. Zhang XD,Chen YR, Ge L, et al. Features of stroke in Chinese diabetes patients: a hospital-based study[J]. J Int Med Res, 2007; 35(4) :540-546.
20. Ortega-Casarrubios MA, Fuentes B, San Jos(?) B, et al. Influence of previous diagnosis of diabetes mellitus in the stroke severity and in-hospital outcome in acute cerebral infarction[J]. Neurologia, 2007; 22(7): 426-433.
21. Pinto A,Tuttolomondo A,Di Raimondo D, et al.A case control study between diabetic and non-diabetic subjects with ischemic stroke[J]. Int Angiol, 2007; 26(1):26-32.
22. Tuttolomondo A, Pinto A, Salemi G, et al.Diabetic and non-diabetic ubjects with ischemic stroke:Differences, subtype distribution and outcome[J]. Nutrition, Metabolism and Cardiovascular Diseases, 2008; 18(2): 152-157.
23. Plengvidhya N, Leelawatana R, Pratipanawatr T, et al. Thailand diabetes registry project: prevalence and risk factors of stroke in thai diabetic patients[J]. J Med Assoc Thai, 2006; 89(Suppl. 1):S49-S53.
24. Matz K, Keresztes K, Tatschl C, et al. Disorders of glucose metabolism in acute stroke patients. An underrecognised problem[J]. Diabetes Care, 2006; 29(4):792-797.
25. Smajlovic D, Salihovic D, Ibrahimagic 0, et al. Stroke in patients with diabetes mellitus:a hospital based study[J].Med Arh, 2006 ; 60(6 Suppl. 2):S63-S65.
26. Khealani BA, Syed NA, Maken S, et al. Predictors of ischemic versus hemorrhagic strokes in hypertensive patients[J]. J Coll Physicians Surg Pak, 2005; 15(1):22-25.
27. Stollberger C, Exner I, Finsterer J, et al. Stroke in diabetic and non-diabetic patients:course and prognostic value of admission serum glucose[J]. Ann Med, 2005; 37 (5):357-364.
28. Zodpey SP, Tiwari RR.A risk scoring system for prediction of haemorrhagic stroke[J]. Indian J Public Health, 2005; 49(4):218-222.
29. Feldmann E, Broderick JP, Kernan WN, et al.Major risk factors for intracerebral hemorrhage in the young are modifiable[J]. Stroke, 2005; 36(9): 1881-1885.
30. Herzig RI, Vlachova JM, Gabrys MD, et al. Occurrence of diabetes mellitus in spontaneous intracerebral hemorrhage[J]. Acta Diabetol, 2007; 44(4): 201-207.
31. Arboix A, Massons J, Garcia-Eroles L, et al. Diabetes is an independent risk factor for in-hospital mortality from acute spontaneous intracerebral hemorrhage[J]. DIABETES CARE,2006; 23(10):1527-1532 .
32. SaccoRL, Adams R, Albers G, et al. Guidelines for prevention of stroke in patients with ischemic stroke or transient ischemic attack: A statement for healthcare professionals from the American Heart Association/American Stroke Association Council on Stroke: Cosponsored by the Council on Cardiovascular Radiology and Intervention: the American Academy of Neurology affirms the value of this guideline[J]. Stroke, 2006; 37(2):577-617.
33. Dalen JE. Aspirin to prevent heart attack and stroke: What' s the right dose?[J]. Am J Med, 2006; 119(3):198-202.
34. He J, Whelton PK, Vu B, et al. Aspirin and risk of hemorrhagic stroke: Ameta-analysis of randomized controlled trials[J]. JAMA, 1998; 280(22): 1930-1935.
35. Hallas J, Dall M, Andries A, et al. Use of single and combined antithrombotic therapy and risk of serious upper gastrointestinal bleeding: Population based case-control study[J]. BMJ, 2006; 333(7571): 726-728.
36. CAPRIE Steering Committee. A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE)[J]. Lancet, 1996; 348 (9038) :1329-1339.
37. Halkes PH, Van Gijn J, Kappelle LJ, et al. Aspirin plus dipyridamole versus aspirin alone after cerebral ischaemia of arterial origin (ESPRIT):Randomised controlled trial[J].Lancet, 2006; 367(9523): 1665-1673.
38. Likosky DJ, Lee K, Brown DM, et al.Evidence-based medicine: Review of guidelines and trials in the prevention of secondary stroke[J].J HOSPITAL MEDICINE, 2008; 3 (Suppl.4) :S6-S19.
39. Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. UK Prospective Diabetes Study Group[J]. BMJ, 1998; 317(7160):703-713.
40. Amarenco P, Bogousslavsky J, Callahan A, et al. High-dose atorvastatin after stroke or transient ischemic attack[J]. N Engl J Med, 2006; 355(6) : 549-559.
41. Bokura H, Kobayashi S, Yamaguchi S, et al. Clinical characteristics and prognosis in stroke patients with diabetes mellitus:retrospective evaluation using the Japanese Standard Stroke Registry database (JSSRS) [J]. Brain and Nerve, 2006; 58(2):135-139.
42.Hacke W,Albers G,Al-Rawi Y,et al.The Desmoteplase in Acute Ischemic Stroke Trial (DIAS):A phaseⅡMRI-based 9-hour window acute stroke thrombolysis trial with intravenous desmoteplase[J].Stroke,2005;36(1):66-73.