S100A4蛋白对运动氧化应激致心血管内皮细胞凋亡的分子调控
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摘要
人体进行力竭运动时,会产生大量氧自由基,诱发心血管内皮细胞凋亡,导致心血管疾病的发生。而诱发心血管内皮细胞损伤的因素很多,其中氧化应激在心血管疾病的发生中起着重要作用。S100蛋白家族是一组结构与功能相似的低分子量钙结合蛋白,具有组织分布特异性。不同S100蛋白具有不同的生物学活性,其细胞内定位及表达模式不同,且可形成同源或异源二聚体,在细胞增殖、分化、肌肉收缩、基因表达、分泌及细胞凋亡中发挥着重要作用,其功能异常亦可导致不同的疾病。S100A4蛋白是S100家族成员之一,人们对它的研究主要集中在肿瘤学领域,其作为促肿瘤转移因子与肿瘤的发生、发展及预后相关。研究表明S100A4蛋白在心血管内皮中有表达,具有促血管生成作用。因此,我们推测S100A4蛋白对防止心血管系统损伤可能起到关键性的保护作用。近来研究表明,在肿瘤细胞增殖过程中,S100A4与P53蛋白的C末端区域结合,影响P53的亚细胞定位及转录活性,从而对细胞凋亡过程产生影响。但S100A4蛋白作为心脏损伤潜在的保护因子,在力竭运动氧化应激心血管内皮细胞凋亡中的调控机制尚不清楚。p53作为细胞凋亡的重要介导因子,S100A4蛋白可以与野生型p53蛋白的C末端的调控区结合,影响其功能。那么S100A4蛋白是否参与了心血管内皮细胞凋亡通路的调节?S100A4蛋白在p53蛋白凋亡途径中发挥作用的机制是什么?有待研究证实。那么在力竭运动氧化应激损伤的内皮细胞凋亡通路中,S100A4蛋白是否通过影响p53蛋白进出核及其转录活性,调控细胞凋亡?目前还未见报道。因此,本课题针对这些问题,通过建立大鼠力竭运动氧化应激心血管内皮细胞整体动物模型和体外培养心血管内皮细胞氧化应激损伤模型,观察S100A4蛋白在力竭运动损伤心血管内皮细胞中的表达变化规律;构建S100A4蛋白表达载体,表达并纯化S100A4蛋白。通过体外干预的方法,观察S100A4蛋白通过p53途径对氧化应激心血管内皮细胞凋亡的影响,初步阐明S100A4蛋白对心血管内皮细胞凋亡的调控机制,为研究心血管内皮损伤防护新措施及开发新制剂开辟新思路。主要研究结果如下:
一、S100A4和p53蛋白在力竭运动氧化应激大鼠心肌组织中的表达分布规律
Wistar大鼠12只,随机分为对照组(Control,CN)和力竭运动损伤组(Exhaustion exercise injury,EI),进行10天连续力竭训练;训练结束后,立刻采集血液和心肌组织样本。结果表明:力竭运动后,大鼠血液中LDH活性(72.13±3.68U/L,P<0.01)显著高于对照组(18.50±2.98U/L),NO含量降低25.15%,而MDA含量升高1.90倍,同时SOD活性下降26.11%。结果表明,力竭运动导致机体损伤,尤其是心血管内皮细胞损伤,提示心血管损伤的主要因素之一是自由基增多造成的氧化应激损伤。HE染色结果揭示,对照组心肌组织结构完整,未见明显病变;力竭运动损伤组主要表现为心肌和血管内皮细胞空泡变性和心肌间质水肿等。在电镜下可见,血管内皮细胞发生线粒体肿胀变形、空泡化,细胞核浓集、边缘化以及凋亡小体出现的典型细胞凋亡特征。由血液生化及病理指标的变化综合判断可知,力竭运动不仅可致心血管氧化应激损伤,还可诱发大鼠心血管内皮细胞凋亡。Western-blot结果可见,CN组心肌组织中S100A4蛋白弱表达,EI组表达量升高;同时检测到野生型P53蛋白仅极少量表达,而EI组表达升高。免疫组织化学方法显示,S100A4和P53蛋白在EI组心肌和血管内皮细胞中均有表达;S100A4表达升高1.68倍,P53蛋白表达升高1.63倍。
二、S100A4蛋白的构建、表达、纯化与鉴定
根据大肠杆菌的密码子偏好性,重新优化并合成了大鼠S100A4基因,并在C端引入了6×His标签,经测序表明,合成的序列和预期结果完全一致,将该序列酶切后连接pBV220载体并转化大肠杆菌E.coli DH5α,经热激诱导后获得了高效可溶性表达,目的表达产物占菌体蛋白的20%以上,Western-blot分析表明,该蛋白是目的产物S100A4蛋白;表达产物破碎后取上清,采用金属螯合层析法进行纯化,最终获得了纯度95%以上的纯化制品,为进一步研究S100A4在氧化应激内皮细胞凋亡中的调控机制奠定了基础。
三、S100A4蛋白对氧化应激内皮细胞凋亡的调控机制研究
采用组织块贴壁法原代培养大鼠主动脉内皮细胞。以浓度为100μmol/L的H2O2干预12h,成功建立内皮细胞氧化应激损伤模型。结果表明,H2O2可导致培养的内皮细胞氧化应激损伤,内皮细胞存活率下降28.67%,并诱发部分内皮细胞凋亡。外源性S100A4蛋白具有逆转氧化应激损伤,提高细胞存活率,抑制内皮细胞凋亡的作用。Western-blot结果表明,内皮细胞加入H2O2后,S100A4和P53蛋白表达水平升高。加入S100A4蛋白进行干预后,内皮细胞胞浆中P53蛋白量升高,而核P53蛋白量降低。加入S100A4蛋白的剂量与核P53蛋白水平降低的程度存在一定关系。提示,S100A4蛋白对氧化应激心血管内皮细胞凋亡的调控,可能是S100A4通过抑制P53入核,从而降低P53促凋亡的作用而发挥保护作用。
综上所述,力竭运动不仅可致心血管氧化应激损伤,还可诱发大鼠心血管内皮细胞凋亡;S100A4和P53蛋白在力竭运动氧化应激损伤大鼠心血管组织中表达升高;成功构建pBV220-S100A4表达载体,获得了高效可溶性表达,最终获得了纯度达95%的蛋白;建立了氧化应激致内皮细胞凋亡细胞模型,结果表明,氧化应激损伤可导致S100A4和P53蛋白表达水平升高。加入S100A4蛋白,可对细胞氧化应激损伤产生保护作用,提高内皮细胞存活率,抑制细胞凋亡;可以影响P53蛋白在氧化应激损伤内皮细胞中的分布,主要是增加P53在胞浆内的聚集,减少P53在核内的的水平,提示S100A4蛋白可阻抑P53由胞浆转入核内,推测这可能是S100A4蛋白对氧化应激内皮细胞凋亡调控机制的重要环节之一。
A lot of oxygen free radicals being produced from human body that carrying out exhaustion exercise could induce apoptosis of cardiovascular endothelial cells. Among the factors that induce endothelial cells injury, oxidative stress plays a key role in inducing cardiovascular diseases. S100 proteins that belong to the family of calcium-binding proteins are a group of calcium-binding proteins that share similar structure and function and low molecular weight. They have distribution specificity of tissue. And different S100 protein has different biological activity and intracellular location and mode of expression. They could form homodimer or heterodimer and play an important role in cell proliferation, cell differentiation, muscle contraction, genetic expression, secretion and apoptosis. Their disfunction also could induce different diseases. S100A4 is a member of the family of S100 proteins, which mainly is centered on oncology area. As a promoted tumor metastasis factor, it is involved in development or growth or prognosis of tumor. Because S100A4 can not only express in cardiovascular endothelial cells, but also promote angiogenesis, we guess that it maybe have key protective effect on cardiovascular system injury. Recently the study revealed that in the course of proliferation of tumor cells , S100A4 binding to the c-terminal area of P53 could affect subcellular localization and transcriptional activity of P53 and apoptosis process of cells. As a potential protective factor of heart injury, the control mechanism of S100A4 on apoptosis of cardiovascular endothelial cells by exhaustion exercise remains elusive. Whether S100A4 is involved in the control of cardiovascular endothelial cell apoptosis and how to affect the apoptosis way of P53 is still to testify for further study. P53 is an important mediate factor of cell apoptosis. S100A4 can bind to the control region of C-terminal of wild type P53 and affect its function. In endothelial cell apoptosis way for exhaustion exercise oxidative stress injury, whether S100A4 affects in-out nucleolus or transcriptional activity of P53 and control apoptosis remains elusive. To explore the change law of S100A4 in cardiovascular endothelial cells injury by exhaustion exercise, we have established both male Wistar rats animal model of cardiovascular endothelial cells injury by exhaustion exercise and cultured vascular endothelial cells oxidative stress injury for H2O2. To construct prokaryotic expression vector for expressing S100A4 protein and establish purification technology of S100A4, the aim of the study is to establish a new way for protecting cardiovascular endothelial cells and to develop a fresh idea for developing a kind of (chemical or pharmaceutical) preparation to observe the effect of S100A4 on apoptosis of cardiovascular endothelial cells induced by H2O2 and to elucidate the control mechanism of S100A4 on apoptosis in vitro .
The following is the mainly results:
1 The expression and distribution of S100A4 and p53 in oxidative stress cardiovascular tissue induced by exhaustion exercise
Male Wistar rats were divided into 2 groups: the control group(CN) and the exhaustion exercise group (EI). After 10 days of loading-increasing treadmill running, the rats in both CN and EI were immediately decapitated for colleting blood sample and cardiovascular tissue sample. The results showed: after exhaustion exercise, lactate dehydrogenase(LDH) activity(72.13±3.68U/L,P<0.01) of rat blood in EI increased significantly as compared with CN(18.50±2.98U/L) ,but NO content of EI declined by 25.15% (P<0.01) when compared with that of CN, which revealed that exhaustion exercise could induce cardiovascular injury, especially vascular endothelial cell(VEC) injury. On the same condition that malondialdehyde(MDA) content increased by 1.90 fold,but superoxide dismutase(SOD) activity declined by 26.11% when compared with that of CN,which showed that an increasing number of oxygen free radicals is a mainly reason that induces oxidative stress injury of cardiovascular endothelial cells.
HE results showed the structure of cardiovascular tissue in CN group is integrity and no pathological changes. And the myocardium and endothelial cells of EI group emerged vacuolar degeneration or interstitial edema et al. Electron microscope results revealed :mitochondria swell,vacuolization ,nucleolus enrich, marginalization,apoptotic body in vascular endothelial cells. According to the results of biochemical index of blood and pathological index, exhaustion exercise not only leads to oxidative stress injury of cardiovascular tissue, but also induces apoptosis of rat cardiovascular endothelial cell.
Western-blot results proved that the expression of wild-type P53 in CN group was little and the expression of wild-type P53 in EI group increased . And the expression of S100A4 in CN group was little and the expression of S100A4 of EI group increased . Immunohistochemistry results also showed that the expression of both S100A4 and wild-type P53 was distributed in both myocardial cells and endothelial cells after exhaustion exercise ; but the expression of S100A4 incresed by 1.68 fold and the expression of wild-type P53 increased by 1.63 fold.
2 Sequence Optimization , High-level Expression , purification and characterization of S100A4 protein
According to the natural S100A4 DNA sequence, the primers were designed based on E.coli codon preference and 6×His tag was introduced. The optimization sequence was amplified by PCR. The BBP sequence was inserted in pEasy-T3 to construct the recombinant cloning vector pEasy-S100A4. After determined by sequence analysis, the S100A4 gene was cloned into expression vector pBV-220 to construct the recombinant expression vector pBV220- S100A4, and was over-expressed in E.coli DH5α. The recombinant protein about 11.5 KDa was showed by SDS-PAGE and was S100A4 protein determined by Western-blot. Fusion protein in supernatant was successfully purified by Ni affinity chromatography. In the final we have successfully produced S100A4 protein(purity coefficient above 95% ).It laid the foundation for further research about the control mechanism of S100A4 in apoptosis of endothelial cells by oxidative stress injury.
3 The control of S100A4 on apoptosis of vascular endothelial cells induced by oxidative stress injury
In the vitro experimental models, the cultured vascular endothelial cells were stimulated by H2O2. The results demonstrated that H2O2 could lead to oxidative stress injury of the cultured vascular endothelial cells and decline the cell survival rate(28.67%) of vascular endothelial cells and induce apoptosis of vascular endothelial cells. Exogenous S100A4 plays an insignificant role in anti-oxidative stress injury and increasing the cell survival rate of vascular endothelial cells and decreasing apoptosis of vascular endothelial cells. Western-blot results suggested: the level on of both S100A4 and P53 increased after oxidative stress injury of vascular endothelial cells induced by H2O2; exogenous S100A4 increased the kytoplasm level on of P53 in endothelial cells and decreased the nuclear level of P53 in endothelial cells; Different dose of exogenous S100A4 declined the different nuclear level of P53 in endothelial cells. We concluded that the control of S100A4 on apoptosis of endothelial cells induced by oxidative stress injury depended on that S100A4 restrained P53 into cell nucleus and declined the effect of P53 on promoting apoptosis of cells.
Take together, these data support that the oxidative stress injury and apoptosis of rat cardiovascular cells was induced by exhaustion exercises. These findings suggested that the expression of both S100A4 and P53 increased in oxidative stress cardiovascular tissue induced by exhaustion exercise. We constructed the recombinant expression vector pBV220- S100A4. Fusion protein in supernatant was successfully purified by Ni affinity chromatography. In the final we have successfully produced S100A4 protein (purity coefficient above 95%). The cell model of apoptosis induced by oxidative stress was established. It is showed that oxidative stress could lead to increasing level of expression of both S100A4 and P53. S100A4 played an important role in anti-oxidative stress endothelial cells injury and promoting survival rate of endothelial cells and decreasing apoptosis of cells. These findings suggested that exogenous S100A4 could affect the distribution of P53 in oxidative stress injury endothelial cells via increasing accumulation of intracytoplasmic P53 and decreasing the intranuclear level of P53. With the dose of S100A4 increasing ,the effect of S100A4 on P53 was proved more significantly, which revealed that S100A4 was able to restrain P53 into cell nucleus. Therefore, we guess that the ability of S100A4 to restrain P53 into cell nucleus perhaps was the one of control mechanism of apoptosis in endothelial cell injury induced by oxidative stress.
一、S100A4和p53蛋白在力竭运动氧化应激大鼠心肌组织中的表达分布规律
Wistar大鼠12只,随机分为对照组(Control,CN)和力竭运动损伤组(Exhaustion exercise injury,EI),进行10天连续力竭训练;训练结束后,立刻采集血液和心肌组织样本。结果表明:力竭运动后,大鼠血液中LDH活性(72.13±3.68U/L,P<0.01)显著高于对照组(18.50±2.98U/L),NO含量降低25.15%,而MDA含量升高1.90倍,同时SOD活性下降26.11%。结果表明,力竭运动导致机体损伤,尤其是心血管内皮细胞损伤,提示心血管损伤的主要因素之一是自由基增多造成的氧化应激损伤。HE染色结果揭示,对照组心肌组织结构完整,未见明显病变;力竭运动损伤组主要表现为心肌和血管内皮细胞空泡变性和心肌间质水肿等。在电镜下可见,血管内皮细胞发生线粒体肿胀变形、空泡化,细胞核浓集、边缘化以及凋亡小体出现的典型细胞凋亡特征。由血液生化及病理指标的变化综合判断可知,力竭运动不仅可致心血管氧化应激损伤,还可诱发大鼠心血管内皮细胞凋亡。Western-blot结果可见,CN组心肌组织中S100A4蛋白弱表达,EI组表达量升高;同时检测到野生型P53蛋白仅极少量表达,而EI组表达升高。免疫组织化学方法显示,S100A4和P53蛋白在EI组心肌和血管内皮细胞中均有表达;S100A4表达升高1.68倍,P53蛋白表达升高1.63倍。
二、S100A4蛋白的构建、表达、纯化与鉴定
根据大肠杆菌的密码子偏好性,重新优化并合成了大鼠S100A4基因,并在C端引入了6×His标签,经测序表明,合成的序列和预期结果完全一致,将该序列酶切后连接pBV220载体并转化大肠杆菌E.coli DH5α,经热激诱导后获得了高效可溶性表达,目的表达产物占菌体蛋白的20%以上,Western-blot分析表明,该蛋白是目的产物S100A4蛋白;表达产物破碎后取上清,采用金属螯合层析法进行纯化,最终获得了纯度95%以上的纯化制品,为进一步研究S100A4在氧化应激内皮细胞凋亡中的调控机制奠定了基础。
三、S100A4蛋白对氧化应激内皮细胞凋亡的调控机制研究
采用组织块贴壁法原代培养大鼠主动脉内皮细胞。以浓度为100μmol/L的H2O2干预12h,成功建立内皮细胞氧化应激损伤模型。结果表明,H2O2可导致培养的内皮细胞氧化应激损伤,内皮细胞存活率下降28.67%,并诱发部分内皮细胞凋亡。外源性S100A4蛋白具有逆转氧化应激损伤,提高细胞存活率,抑制内皮细胞凋亡的作用。Western-blot结果表明,内皮细胞加入H2O2后,S100A4和P53蛋白表达水平升高。加入S100A4蛋白进行干预后,内皮细胞胞浆中P53蛋白量升高,而核P53蛋白量降低。加入S100A4蛋白的剂量与核P53蛋白水平降低的程度存在一定关系。提示,S100A4蛋白对氧化应激心血管内皮细胞凋亡的调控,可能是S100A4通过抑制P53入核,从而降低P53促凋亡的作用而发挥保护作用。
综上所述,力竭运动不仅可致心血管氧化应激损伤,还可诱发大鼠心血管内皮细胞凋亡;S100A4和P53蛋白在力竭运动氧化应激损伤大鼠心血管组织中表达升高;成功构建pBV220-S100A4表达载体,获得了高效可溶性表达,最终获得了纯度达95%的蛋白;建立了氧化应激致内皮细胞凋亡细胞模型,结果表明,氧化应激损伤可导致S100A4和P53蛋白表达水平升高。加入S100A4蛋白,可对细胞氧化应激损伤产生保护作用,提高内皮细胞存活率,抑制细胞凋亡;可以影响P53蛋白在氧化应激损伤内皮细胞中的分布,主要是增加P53在胞浆内的聚集,减少P53在核内的的水平,提示S100A4蛋白可阻抑P53由胞浆转入核内,推测这可能是S100A4蛋白对氧化应激内皮细胞凋亡调控机制的重要环节之一。
A lot of oxygen free radicals being produced from human body that carrying out exhaustion exercise could induce apoptosis of cardiovascular endothelial cells. Among the factors that induce endothelial cells injury, oxidative stress plays a key role in inducing cardiovascular diseases. S100 proteins that belong to the family of calcium-binding proteins are a group of calcium-binding proteins that share similar structure and function and low molecular weight. They have distribution specificity of tissue. And different S100 protein has different biological activity and intracellular location and mode of expression. They could form homodimer or heterodimer and play an important role in cell proliferation, cell differentiation, muscle contraction, genetic expression, secretion and apoptosis. Their disfunction also could induce different diseases. S100A4 is a member of the family of S100 proteins, which mainly is centered on oncology area. As a promoted tumor metastasis factor, it is involved in development or growth or prognosis of tumor. Because S100A4 can not only express in cardiovascular endothelial cells, but also promote angiogenesis, we guess that it maybe have key protective effect on cardiovascular system injury. Recently the study revealed that in the course of proliferation of tumor cells , S100A4 binding to the c-terminal area of P53 could affect subcellular localization and transcriptional activity of P53 and apoptosis process of cells. As a potential protective factor of heart injury, the control mechanism of S100A4 on apoptosis of cardiovascular endothelial cells by exhaustion exercise remains elusive. Whether S100A4 is involved in the control of cardiovascular endothelial cell apoptosis and how to affect the apoptosis way of P53 is still to testify for further study. P53 is an important mediate factor of cell apoptosis. S100A4 can bind to the control region of C-terminal of wild type P53 and affect its function. In endothelial cell apoptosis way for exhaustion exercise oxidative stress injury, whether S100A4 affects in-out nucleolus or transcriptional activity of P53 and control apoptosis remains elusive. To explore the change law of S100A4 in cardiovascular endothelial cells injury by exhaustion exercise, we have established both male Wistar rats animal model of cardiovascular endothelial cells injury by exhaustion exercise and cultured vascular endothelial cells oxidative stress injury for H2O2. To construct prokaryotic expression vector for expressing S100A4 protein and establish purification technology of S100A4, the aim of the study is to establish a new way for protecting cardiovascular endothelial cells and to develop a fresh idea for developing a kind of (chemical or pharmaceutical) preparation to observe the effect of S100A4 on apoptosis of cardiovascular endothelial cells induced by H2O2 and to elucidate the control mechanism of S100A4 on apoptosis in vitro .
The following is the mainly results:
1 The expression and distribution of S100A4 and p53 in oxidative stress cardiovascular tissue induced by exhaustion exercise
Male Wistar rats were divided into 2 groups: the control group(CN) and the exhaustion exercise group (EI). After 10 days of loading-increasing treadmill running, the rats in both CN and EI were immediately decapitated for colleting blood sample and cardiovascular tissue sample. The results showed: after exhaustion exercise, lactate dehydrogenase(LDH) activity(72.13±3.68U/L,P<0.01) of rat blood in EI increased significantly as compared with CN(18.50±2.98U/L) ,but NO content of EI declined by 25.15% (P<0.01) when compared with that of CN, which revealed that exhaustion exercise could induce cardiovascular injury, especially vascular endothelial cell(VEC) injury. On the same condition that malondialdehyde(MDA) content increased by 1.90 fold,but superoxide dismutase(SOD) activity declined by 26.11% when compared with that of CN,which showed that an increasing number of oxygen free radicals is a mainly reason that induces oxidative stress injury of cardiovascular endothelial cells.
HE results showed the structure of cardiovascular tissue in CN group is integrity and no pathological changes. And the myocardium and endothelial cells of EI group emerged vacuolar degeneration or interstitial edema et al. Electron microscope results revealed :mitochondria swell,vacuolization ,nucleolus enrich, marginalization,apoptotic body in vascular endothelial cells. According to the results of biochemical index of blood and pathological index, exhaustion exercise not only leads to oxidative stress injury of cardiovascular tissue, but also induces apoptosis of rat cardiovascular endothelial cell.
Western-blot results proved that the expression of wild-type P53 in CN group was little and the expression of wild-type P53 in EI group increased . And the expression of S100A4 in CN group was little and the expression of S100A4 of EI group increased . Immunohistochemistry results also showed that the expression of both S100A4 and wild-type P53 was distributed in both myocardial cells and endothelial cells after exhaustion exercise ; but the expression of S100A4 incresed by 1.68 fold and the expression of wild-type P53 increased by 1.63 fold.
2 Sequence Optimization , High-level Expression , purification and characterization of S100A4 protein
According to the natural S100A4 DNA sequence, the primers were designed based on E.coli codon preference and 6×His tag was introduced. The optimization sequence was amplified by PCR. The BBP sequence was inserted in pEasy-T3 to construct the recombinant cloning vector pEasy-S100A4. After determined by sequence analysis, the S100A4 gene was cloned into expression vector pBV-220 to construct the recombinant expression vector pBV220- S100A4, and was over-expressed in E.coli DH5α. The recombinant protein about 11.5 KDa was showed by SDS-PAGE and was S100A4 protein determined by Western-blot. Fusion protein in supernatant was successfully purified by Ni affinity chromatography. In the final we have successfully produced S100A4 protein(purity coefficient above 95% ).It laid the foundation for further research about the control mechanism of S100A4 in apoptosis of endothelial cells by oxidative stress injury.
3 The control of S100A4 on apoptosis of vascular endothelial cells induced by oxidative stress injury
In the vitro experimental models, the cultured vascular endothelial cells were stimulated by H2O2. The results demonstrated that H2O2 could lead to oxidative stress injury of the cultured vascular endothelial cells and decline the cell survival rate(28.67%) of vascular endothelial cells and induce apoptosis of vascular endothelial cells. Exogenous S100A4 plays an insignificant role in anti-oxidative stress injury and increasing the cell survival rate of vascular endothelial cells and decreasing apoptosis of vascular endothelial cells. Western-blot results suggested: the level on of both S100A4 and P53 increased after oxidative stress injury of vascular endothelial cells induced by H2O2; exogenous S100A4 increased the kytoplasm level on of P53 in endothelial cells and decreased the nuclear level of P53 in endothelial cells; Different dose of exogenous S100A4 declined the different nuclear level of P53 in endothelial cells. We concluded that the control of S100A4 on apoptosis of endothelial cells induced by oxidative stress injury depended on that S100A4 restrained P53 into cell nucleus and declined the effect of P53 on promoting apoptosis of cells.
Take together, these data support that the oxidative stress injury and apoptosis of rat cardiovascular cells was induced by exhaustion exercises. These findings suggested that the expression of both S100A4 and P53 increased in oxidative stress cardiovascular tissue induced by exhaustion exercise. We constructed the recombinant expression vector pBV220- S100A4. Fusion protein in supernatant was successfully purified by Ni affinity chromatography. In the final we have successfully produced S100A4 protein (purity coefficient above 95%). The cell model of apoptosis induced by oxidative stress was established. It is showed that oxidative stress could lead to increasing level of expression of both S100A4 and P53. S100A4 played an important role in anti-oxidative stress endothelial cells injury and promoting survival rate of endothelial cells and decreasing apoptosis of cells. These findings suggested that exogenous S100A4 could affect the distribution of P53 in oxidative stress injury endothelial cells via increasing accumulation of intracytoplasmic P53 and decreasing the intranuclear level of P53. With the dose of S100A4 increasing ,the effect of S100A4 on P53 was proved more significantly, which revealed that S100A4 was able to restrain P53 into cell nucleus. Therefore, we guess that the ability of S100A4 to restrain P53 into cell nucleus perhaps was the one of control mechanism of apoptosis in endothelial cell injury induced by oxidative stress.
引文
[1] Schmieder RE. Endothelial dysfunction : how can one intervene at the beginning of the cardiovascular continuum ?[J ] . J Hypertens Suppl ,2006 ,24 (2) :31
[2] Callaghan MJ , Ceradini DJ , Gurtner GC. Hyperglycemia-induced reactive oxygen species and impaired endothelial progenitor cell function [J ] . Antioxid Redox signal ,2005,7(11～12) :1476
[3] Navrro-Arevalo A,Canavate C, Sanchez-del-Pino MJ. Myocardial and skeletal muscle aging and changes in oxidative stress in relationship to rigorous exercise training[J]. Mech Ageing Dev. 1999,108(3):207-17.
[4]邱雅慧。血管内皮细胞的功能以及损伤修复与动脉粥样硬化。中国组织工程研究与临床康复,2007,11(10):1927-1929.
[5] Crimi E, Ignarro LJ, Napoli C. Microcirculation and oxidative stress[J]. Free Radic Res.2007 ,41(12):1364-75.
[6]任德成,杜冠华。血管内皮细胞的氧化性损伤机理。心血管病学进展,2002,23(4):235-238.
[7]钱呈睿,葛海良,王颖。p53转录非依赖活性介导细胞凋亡。生命科学,2007 ,19(3):326-329.
[8] Tarabykina S, Griffiths T R L, Tulchinsky J K, et al. Metastasis- associated protein S100A4: Spotlight on its role in cell migration[J]. Current cancer drug targets. 2007,7(3):217-228.
[9] Moroz OV, Antson AA, Murshudov GN,et al. The three dimensional structure of human S100A12[J]. Acta Crystallogr D Biol Crystallogr, 2001,57:20-29.
[10] Ridinger K, Schafer BW, Durussel I, et al. S100A13 biochemical characterization and subcellular localization in different cell lines[J]. J Biol Chem, 2000,275(12):8686-94.
[11] Mazzuccheli L. Protein S100A4: too long overlooked by pathologists?[J]. Am J Pathol, 2002,160:7-13.
[12] Donato R. S100:a multigenic family of calcium– modulated proteins of the EF-hand type with intracellular and extracelluar functions roles[J]. Int J Biochem Cell Biol,2001;33(7):637～668.
[13] Garrett SC, Varney KM, Weber DJ, Bresnick AR. S100A4, a mediator ofmetastasis[J]. J Biol Chem, 2006,281:677–80.
[14]Ambartsumian N, Grigorian M, Lukanidin E. Genetically modified mouse models to study the role of metasasis-promoting S100A4(mts1) protein in metastatic mammary cancer[J].J Dairy Res,2005,72(S1):27-33.
[15]Schmidt Hansen B, Klingelhofer J,Grum-Schwensen B,et al.Functional significance of metastasis-including S100A4(Mts1) in tumor-stroma interplay[J].J Biol Chem,2004,279:24498-24504.
[16] Mikael Schneider, Sawa Kostin,Claes C,et al. S100A4 is regulated in injured myocardium and promotes growth and survival of cardiac myocytes[J].Cardiovasc Res,2007,75;40-50.
[17]Stary M, Schneider M, Sheikh SP, Weitzer G. Parietal endoderm secreted S100A4 promotes early cardiomyogenesis in embryoid bodies[J]. Biochem Biophys Res Commun, 2006,343:555–63.
[18] Str?m CC, Kruh?ffer M, Knudsen S, Stensgaard-Hansen F, Jonassen TEN, ?rntoft TF, et al. Identification of a core set of genes that signifies pathways underlying cardiac hypertrophy[J]. Compar Funct Genom ,2004,5:459–70.
[19]Grigorian M ,Andresen S, Tulchinsky E, et al. Tumor suppressor P53 protein is a new target for the metastasis-associated Mts1/S100A4 protein: functional consequences of their interaction[J].J Biol Chem,2001,276:22699-22708.
[20]Hayashih. Pathogenesis and the role of Ca2+ overload during myocardial ischemia/reperfusion[J].Nagoya Med Sci, 2000,63(3-4):91-98.
[21]Lukanidian EM, Georgiev GP. Metastasis-related mtas1 gene[J]. Curr Top Microbiol Immunol,1996,213:171-195.
[22]Sherbet GV, Lakshmi MS.S100A4(Mts1) calcium binding protein in cancer growth invasion and metastasis[J]. Anticancer Res,1998,18(4A):2415-2421.
[23] Taylor S, Herrington S, Prime W, et al. S100A4 (p9Ka) protein in colon carcinoma and liver metastases: association with carcinoma cells and T-lymphocytes[J].Br J Cancer, 2002,86(3):409-416.
[24]Bedford TG, Tipton CM, Wison NC, et al. Maximal oxygen consumption of rats and its changes with various experimental procedures. J Appl Physiol, 1979, 47(6):1278-1283.
[25]田野,高铁群.大鼠运动性疲劳模型的建立.北京体育大学学报,1995,18(4):49-53.
[26]王伯沄,李玉松,黄高昇,张远强。病理学技术[M],第1版.北京:人民卫生出版社,2006:73.
[27] DemenicsE PG Excitatory amino acid release and free radical formation may operate in the fenesis of ischem in induced neuronal damage[J]. Neurosci 1999;10(3):1035
[28]Morooka H, Hirotsune N, Wani T, et al. Histochemical demolilration of free radicals (H2O2)in ischemic brain edema and Protective effects of human recombinant Superoxide dismutase on ischemic neuronal damage[J]. Acta Neurochir SuPPI(Wien)1994:60:307-309
[29] Marenholz I,Heizmann C W,Fritz G. S100 proteins in mouse and man: from evolution to function and pathology(including an update of the nomenclature) [J]. Biochem Biophys Res Commun, 2004, 322(pt4):1111-1122.
[30] Santamaria-Kistel L,Rintala-Dempsey A C,Shaw G S. Calcium-dependent and–independent interactions of the S100 protein family[J]. Biochem, 2006, 396(4):201-214.
[31] Xinxin Gao, Peggy Yo, Andrew Keith, Timothy J.Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-?delity assembly of longer gene sequences[J]. Nucleic Acids Research, 2003, 31( 22):143-154.
[32]Purification and renaturation of recombinant proteins produced in Escherichia coli as inclusion bodies[J]. Application Note 18-1112-33, Amersham Pharmacia Biotech.
[33]Colangeli, R, Heijbel, A., Williams, A.M., et al. purification of lipopolysaccharide-free polyhistidine-tagged recombinant antigens of Myobacterium tuberculosis[J]. J Chromatography , 714 (1998) 223–235.
[34] Rapid and efficient purificaton and refolding of a (His)6-tagged recombinant protein produced in E. coli as inclusion bodies[J]. Application Note 18-1134-37,Amersham Pharmacia Biotech.
[35] Kreisel D,Krupnick AS,Szeto WY,et al.A simple method for culturing mouse vascular endothelium[J].J Immunol Methods,2001,254(1~2):31-45.
[36]章静波。组织和细胞培养技术[M],第1版.北京:人民卫生出版社,2004:115-116.
[37]姚泰.生理学[M].第6版.北京:人民卫生出版社,2004:57-59.
[38] Jungersten L.Ambfing A.Wall B.et al Both physical fitness and acute exercise regulate nitric oxide formation in healthy humans[J].J Appl Physiol 1997: 82(3):760-4.
[39] Lubov T,Marmor A.Gorenberg M Endothelin release:a marker for the sevenb of exercise-induced ischemia lnt[J]. J Cardiol 2001:79(1):19-24.
[40]赵敬国王福文.力竭性运动后不同时相大鼠心肌形态结构的改变观察[J]中国运动医学杂志,2001.20(3):316-7.
[41]丁树哲.运动中内源性自由基对大鼠心肌线粒体膜分子动力学的影响[J].生物化学和生物物理学报.1991(23):305.
[42]田野.运动性骨骼肌结构、机能变化的机制研究-Ⅱ。力竭运动对线粒体钙代谢水平的影响[J].中国运动医学杂志,1993(12):31-33.
[43]刘丽萍.游泳训练对大鼠肝组织自由基代谢及肝脏超微结构的影响[J].中国运动医学杂志,1998.17(2):121-123.
[44]常波.力竭性游泳对大鼠红细胞膜脂质过氧化、钠泵和红细胞变形性的影响[J].沈阳体育学院学报,1998(4):1.
[45] Yonish-Rouach, D Grunwald, S Wilder, et al. p53-mediated cell death: relationship to cell cycle control[J]. Mol Cell Biol. 1993; 13(3): 1415-1423
[46] Sakamoto T, Repasky WT, Uchida K. Modulation of cell death pathways to apoptosis and necrosis of H202-treated rat thymocytes by lipocortin1[J].Biochem Biophys Res Comunun.1996;220:643-647
[47] Mosman T. Rapid colorimeteic assay for cellur growth and survival:application to proliferation and cytotoxicity assay[J]. J Immunol Methods.1983;65(1):55-63.
[48]Raij L. Hypertension, endothelium and cardiovascular risk factors[J].Am J Med 1990 (supple 2A):13
[49]Smith EJ, Rothman M T.Antiproliferative coatings for the treatment of coronary heart disease: what are the targets and which are the tools?[J]. J Interv Cardiol 2003: 16:475-483.
[50] Lavin M F, Gueven N. The complexity of p53 stabilization and activation[J]. CellDeath Differ. 2006,13(6):941-950.
[51] Mueller A, Schafer B W, Ferrai S, et al. The calcium-binding protein S100A2 interact with p53 and modulates its transcriptional activity[J]. J Biol Chem.2005,280(32):29186-29193.
[52] Grigorian M, Andresen S,Tulchinsky E, et al. Tumor suppressor p53 protein is a new target for the metastasis-associated Mts1/S100A4 protein: functional consequences of their interaction[J].J Biol Chem. 2001,276(25):22699-22708.
[53] Tarabykina S, Griffiths T R L, Tulchinsky J K, et al. Metastasis- associated protein S100A4: Spotlight on its role in cell migration[J]. Current cancer drug targets. 2007,7(3):217-228.
[54] Dulyaninova N G, Malashkevich V N, Almo S C, et al. Regulation of myosin-ⅡA assembly and Mts1 binding by heavy chain phosphorylation[J]. Biochemistry. 2005,44(8):6867-6876.
[55] Fernandez-Fernandez M R, Veprintsev D B. Fersht A R. Proteins of the S100 family regulate the oligomerization of p53 tumor suppressor[J]. Proc Natl Acad Sci USA.2005,102(13):4735- 4740.
[56] Chen H, Fernig D G, Rudland PS, et al. Binding to intracellular targets of the metastasis- inducing protein, S100A4(p9Ka) [J]. Biochem Biophys Res Commun.2001,286(5):1212-1217.
[2] Callaghan MJ , Ceradini DJ , Gurtner GC. Hyperglycemia-induced reactive oxygen species and impaired endothelial progenitor cell function [J ] . Antioxid Redox signal ,2005,7(11～12) :1476
[3] Navrro-Arevalo A,Canavate C, Sanchez-del-Pino MJ. Myocardial and skeletal muscle aging and changes in oxidative stress in relationship to rigorous exercise training[J]. Mech Ageing Dev. 1999,108(3):207-17.
[4]邱雅慧。血管内皮细胞的功能以及损伤修复与动脉粥样硬化。中国组织工程研究与临床康复,2007,11(10):1927-1929.
[5] Crimi E, Ignarro LJ, Napoli C. Microcirculation and oxidative stress[J]. Free Radic Res.2007 ,41(12):1364-75.
[6]任德成,杜冠华。血管内皮细胞的氧化性损伤机理。心血管病学进展,2002,23(4):235-238.
[7]钱呈睿,葛海良,王颖。p53转录非依赖活性介导细胞凋亡。生命科学,2007 ,19(3):326-329.
[8] Tarabykina S, Griffiths T R L, Tulchinsky J K, et al. Metastasis- associated protein S100A4: Spotlight on its role in cell migration[J]. Current cancer drug targets. 2007,7(3):217-228.
[9] Moroz OV, Antson AA, Murshudov GN,et al. The three dimensional structure of human S100A12[J]. Acta Crystallogr D Biol Crystallogr, 2001,57:20-29.
[10] Ridinger K, Schafer BW, Durussel I, et al. S100A13 biochemical characterization and subcellular localization in different cell lines[J]. J Biol Chem, 2000,275(12):8686-94.
[11] Mazzuccheli L. Protein S100A4: too long overlooked by pathologists?[J]. Am J Pathol, 2002,160:7-13.
[12] Donato R. S100:a multigenic family of calcium– modulated proteins of the EF-hand type with intracellular and extracelluar functions roles[J]. Int J Biochem Cell Biol,2001;33(7):637～668.
[13] Garrett SC, Varney KM, Weber DJ, Bresnick AR. S100A4, a mediator ofmetastasis[J]. J Biol Chem, 2006,281:677–80.
[14]Ambartsumian N, Grigorian M, Lukanidin E. Genetically modified mouse models to study the role of metasasis-promoting S100A4(mts1) protein in metastatic mammary cancer[J].J Dairy Res,2005,72(S1):27-33.
[15]Schmidt Hansen B, Klingelhofer J,Grum-Schwensen B,et al.Functional significance of metastasis-including S100A4(Mts1) in tumor-stroma interplay[J].J Biol Chem,2004,279:24498-24504.
[16] Mikael Schneider, Sawa Kostin,Claes C,et al. S100A4 is regulated in injured myocardium and promotes growth and survival of cardiac myocytes[J].Cardiovasc Res,2007,75;40-50.
[17]Stary M, Schneider M, Sheikh SP, Weitzer G. Parietal endoderm secreted S100A4 promotes early cardiomyogenesis in embryoid bodies[J]. Biochem Biophys Res Commun, 2006,343:555–63.
[18] Str?m CC, Kruh?ffer M, Knudsen S, Stensgaard-Hansen F, Jonassen TEN, ?rntoft TF, et al. Identification of a core set of genes that signifies pathways underlying cardiac hypertrophy[J]. Compar Funct Genom ,2004,5:459–70.
[19]Grigorian M ,Andresen S, Tulchinsky E, et al. Tumor suppressor P53 protein is a new target for the metastasis-associated Mts1/S100A4 protein: functional consequences of their interaction[J].J Biol Chem,2001,276:22699-22708.
[20]Hayashih. Pathogenesis and the role of Ca2+ overload during myocardial ischemia/reperfusion[J].Nagoya Med Sci, 2000,63(3-4):91-98.
[21]Lukanidian EM, Georgiev GP. Metastasis-related mtas1 gene[J]. Curr Top Microbiol Immunol,1996,213:171-195.
[22]Sherbet GV, Lakshmi MS.S100A4(Mts1) calcium binding protein in cancer growth invasion and metastasis[J]. Anticancer Res,1998,18(4A):2415-2421.
[23] Taylor S, Herrington S, Prime W, et al. S100A4 (p9Ka) protein in colon carcinoma and liver metastases: association with carcinoma cells and T-lymphocytes[J].Br J Cancer, 2002,86(3):409-416.
[24]Bedford TG, Tipton CM, Wison NC, et al. Maximal oxygen consumption of rats and its changes with various experimental procedures. J Appl Physiol, 1979, 47(6):1278-1283.
[25]田野,高铁群.大鼠运动性疲劳模型的建立.北京体育大学学报,1995,18(4):49-53.
[26]王伯沄,李玉松,黄高昇,张远强。病理学技术[M],第1版.北京:人民卫生出版社,2006:73.
[27] DemenicsE PG Excitatory amino acid release and free radical formation may operate in the fenesis of ischem in induced neuronal damage[J]. Neurosci 1999;10(3):1035
[28]Morooka H, Hirotsune N, Wani T, et al. Histochemical demolilration of free radicals (H2O2)in ischemic brain edema and Protective effects of human recombinant Superoxide dismutase on ischemic neuronal damage[J]. Acta Neurochir SuPPI(Wien)1994:60:307-309
[29] Marenholz I,Heizmann C W,Fritz G. S100 proteins in mouse and man: from evolution to function and pathology(including an update of the nomenclature) [J]. Biochem Biophys Res Commun, 2004, 322(pt4):1111-1122.
[30] Santamaria-Kistel L,Rintala-Dempsey A C,Shaw G S. Calcium-dependent and–independent interactions of the S100 protein family[J]. Biochem, 2006, 396(4):201-214.
[31] Xinxin Gao, Peggy Yo, Andrew Keith, Timothy J.Thermodynamically balanced inside-out (TBIO) PCR-based gene synthesis: a novel method of primer design for high-?delity assembly of longer gene sequences[J]. Nucleic Acids Research, 2003, 31( 22):143-154.
[32]Purification and renaturation of recombinant proteins produced in Escherichia coli as inclusion bodies[J]. Application Note 18-1112-33, Amersham Pharmacia Biotech.
[33]Colangeli, R, Heijbel, A., Williams, A.M., et al. purification of lipopolysaccharide-free polyhistidine-tagged recombinant antigens of Myobacterium tuberculosis[J]. J Chromatography , 714 (1998) 223–235.
[34] Rapid and efficient purificaton and refolding of a (His)6-tagged recombinant protein produced in E. coli as inclusion bodies[J]. Application Note 18-1134-37,Amersham Pharmacia Biotech.
[35] Kreisel D,Krupnick AS,Szeto WY,et al.A simple method for culturing mouse vascular endothelium[J].J Immunol Methods,2001,254(1~2):31-45.
[36]章静波。组织和细胞培养技术[M],第1版.北京:人民卫生出版社,2004:115-116.
[37]姚泰.生理学[M].第6版.北京:人民卫生出版社,2004:57-59.
[38] Jungersten L.Ambfing A.Wall B.et al Both physical fitness and acute exercise regulate nitric oxide formation in healthy humans[J].J Appl Physiol 1997: 82(3):760-4.
[39] Lubov T,Marmor A.Gorenberg M Endothelin release:a marker for the sevenb of exercise-induced ischemia lnt[J]. J Cardiol 2001:79(1):19-24.
[40]赵敬国王福文.力竭性运动后不同时相大鼠心肌形态结构的改变观察[J]中国运动医学杂志,2001.20(3):316-7.
[41]丁树哲.运动中内源性自由基对大鼠心肌线粒体膜分子动力学的影响[J].生物化学和生物物理学报.1991(23):305.
[42]田野.运动性骨骼肌结构、机能变化的机制研究-Ⅱ。力竭运动对线粒体钙代谢水平的影响[J].中国运动医学杂志,1993(12):31-33.
[43]刘丽萍.游泳训练对大鼠肝组织自由基代谢及肝脏超微结构的影响[J].中国运动医学杂志,1998.17(2):121-123.
[44]常波.力竭性游泳对大鼠红细胞膜脂质过氧化、钠泵和红细胞变形性的影响[J].沈阳体育学院学报,1998(4):1.
[45] Yonish-Rouach, D Grunwald, S Wilder, et al. p53-mediated cell death: relationship to cell cycle control[J]. Mol Cell Biol. 1993; 13(3): 1415-1423
[46] Sakamoto T, Repasky WT, Uchida K. Modulation of cell death pathways to apoptosis and necrosis of H202-treated rat thymocytes by lipocortin1[J].Biochem Biophys Res Comunun.1996;220:643-647
[47] Mosman T. Rapid colorimeteic assay for cellur growth and survival:application to proliferation and cytotoxicity assay[J]. J Immunol Methods.1983;65(1):55-63.
[48]Raij L. Hypertension, endothelium and cardiovascular risk factors[J].Am J Med 1990 (supple 2A):13
[49]Smith EJ, Rothman M T.Antiproliferative coatings for the treatment of coronary heart disease: what are the targets and which are the tools?[J]. J Interv Cardiol 2003: 16:475-483.
[50] Lavin M F, Gueven N. The complexity of p53 stabilization and activation[J]. CellDeath Differ. 2006,13(6):941-950.
[51] Mueller A, Schafer B W, Ferrai S, et al. The calcium-binding protein S100A2 interact with p53 and modulates its transcriptional activity[J]. J Biol Chem.2005,280(32):29186-29193.
[52] Grigorian M, Andresen S,Tulchinsky E, et al. Tumor suppressor p53 protein is a new target for the metastasis-associated Mts1/S100A4 protein: functional consequences of their interaction[J].J Biol Chem. 2001,276(25):22699-22708.
[53] Tarabykina S, Griffiths T R L, Tulchinsky J K, et al. Metastasis- associated protein S100A4: Spotlight on its role in cell migration[J]. Current cancer drug targets. 2007,7(3):217-228.
[54] Dulyaninova N G, Malashkevich V N, Almo S C, et al. Regulation of myosin-ⅡA assembly and Mts1 binding by heavy chain phosphorylation[J]. Biochemistry. 2005,44(8):6867-6876.
[55] Fernandez-Fernandez M R, Veprintsev D B. Fersht A R. Proteins of the S100 family regulate the oligomerization of p53 tumor suppressor[J]. Proc Natl Acad Sci USA.2005,102(13):4735- 4740.
[56] Chen H, Fernig D G, Rudland PS, et al. Binding to intracellular targets of the metastasis- inducing protein, S100A4(p9Ka) [J]. Biochem Biophys Res Commun.2001,286(5):1212-1217.
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