【作者】 刘先宝；

【导师】 王建安；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 实验背景:随着经济社会的发展和人民生活水平的提高,心脑血管疾病已经成为人类健康的头号杀手。心肌梗死的发生率、致残率和致死率明显上升,尽管近年来药物治疗和介入治疗得到了快速发展,缺血性心肌病患者的预后有了明显的改善,但药物治疗和介入治疗终归不能修复坏死的心肌。骨髓间充质干细胞(MSCs)具有自我更新能力和横向分化能力,移植到心梗局部后能够分化为内皮细胞、血管平滑肌细胞和心肌细胞样细胞,促进新生血管的形成。大量的基础研究和临床前期研究发现MSCs能够挽救濒死的心肌,减少心梗面积进而改善心肌梗死后的心功能,因此骨髓间充质干细胞在心血管疾病中的应用是近年来研究的热点,成为攻克心血管疾病难题的一种非常有前景的治疗手段。但是,MSCs改善心功能的能力有限,大量的证据表明MSC移植对心功能的改善是轻中度的,大部分研究结果显示MSCs移植对心功能的改善只有5-6%左右,甚至有些临床试验没有观察到心功能的改善,这远远达不到我们的预期,其中一个很重要的原因是绝大多数的细胞移植到体内后在很短的时间内发生死亡,研究报道90%的MSCs在移植后的1天内死亡。因此,找到一种能够提高MSCs生存能力的方法显得尤为迫切。DMOG是一种脯氨酰羟化酶抑制剂,能够抑制细胞内HIF-1α的降解,继而使细胞核内很多基因(如EPO、VEGF、Ang1和Glut-1等)的转录和表达增高,继而发挥其保护性作用。在缺血再灌注心肌损伤和急性心肌梗死的动物模型中发现,DMOG能够发挥降低大鼠的心肌重构和抑制炎症反应等保护作用。在体外的研究中发现,DMOG能够改善肿瘤细胞和神经元在缺血清或缺神经营养因子等恶劣环境下的生存能力。但是,DMOG到底能否改善MSCs的生存能力目前没有报道。实验目的:研究DMOG是否能增强骨髓间充质干细胞(MSCs)的生存能力,并对可能的作用机制进行探讨。实验方法:建立经典的缺血清的凋亡模型,分为正常对照组(Control)、缺血清组(SD)和缺血清DMOG干预组(SD+DMOG)三大组,其中SD+DMOG组又分为100μM、500μM和1000μM三个小组。应用western-blot技术检测DMOG能否降低HIF-1α的在细胞内的降解以及上调其下游Glut-1的表达情况;应用台盼蓝方法来评估DMOG对正常有血清条件培养下的MSCs的毒性作用;通过相差显微镜观察不同组中MSCs形态学上的改变,经缺血清处理后的凋亡细胞体积缩小;应用Hoechst染MSCs的细胞核,通过荧光显微镜观察不同组中MSCs细胞核的改变,凋亡细胞染色质浓聚、细胞核缩小甚至断裂,荧光显微镜下表现为核小而亮和不完整;应用免疫组化对cleaved caspase-3进行染色,阳性细胞定义为凋亡细胞,并使用Hoechst进行染核计算细胞总数,阳性细胞除以总细胞数即为凋亡率;western-blot检测cleaved caspase-3的表达情况,以进一步验证免疫组化染色的准确性,并进一步探讨DMOG对MSCs凋亡保护的时程进行分析,分6h、12h和24h三个时间点;应用台盼蓝方法对MSCs进行染色来检测细胞死亡情况,阳性细胞即为死亡细胞,阳性细胞除以总细胞数即为死亡率。通过比较不同组之间凋亡和死亡指标的变化,说明DMOG对缺血清条件下MSCs的保护作用。对于除HIF-1α通路外其他的可能作用机制,我们检测了经典的影响细胞凋亡的线粒体通路和与细胞生存密切相关的PI3K/Akt通路。应用western-blot技术检测细胞色素C蛋白在线粒体、胞浆中的表达情况,分析在缺血清状况下细胞色素C从线粒体释放到胞浆中(细胞色素C将进一步与Apaf-1和pro-caspase9形成凋亡复合体激活凋亡通路)的情况,探讨线粒体途径中caspase-3依赖性的凋亡通路在DMOG抗凋亡中的作用;应用western-blot技术检测凋亡诱导因子(AIF)在胞浆和核蛋白中的表达情况,分析在缺血清状况下AIF从胞浆转录到细胞核(AIF转录到细胞核后也能够启动细胞凋亡程序)的情况,探讨线粒体途径中非caspase-3依赖性的凋亡通路在DMOG抗凋亡中的作用。应用western-blot技术检测Akt和磷酸化Akt、ERK和磷酸化ERK的表达情况,明确DMOG能否激活PI3K/Akt和/或MEK/ERK通路,然后应用PI3K特异性阻断剂wortmannin阻断PI3K/Akt通路后,观察DMOG的保护作用(重新检测cleaved caspase-3染色和台盼蓝分别评估细胞凋亡和死亡)是否消失,从反面来证明DMOG的保护作用是否通过该通路。实验结果:DMOG在作用浓度范围内,对MSCs没有明显的毒性作用,却能够剂量依赖性地降低MSCs内HIF-1α的降解,上调其下游Glut-1的表达。DMOG能够呈剂量依赖性地改善缺血清后MSCs的形态学上的细胞缩小情况,使Hoechst阳性的细胞核数量明显降低,cleaved caspase-3的表达降低。Western-blot结果进一步确认了免疫染色cleaved caspase-3的结果,并提示从缺血清6h开始,DMOG就有降低凋亡的趋势,到12h和24h时达到了统计学差异。类似地,根据台盼蓝染色结果DMOG能够呈剂量依赖性地降低MSCs经缺血清处理后的细胞死亡,从12h开始到72h均能不同程度地降低细胞死亡,具有显著的统计学意义。以上说明DMOG能够降低缺血清所引起的MSCs凋亡和死亡,并呈剂量依赖性和有一定的作用时程。缺血清能够使细胞色素C从线粒体释放到胞浆的数量增加,而DMOG却能降低这种过程;类似地,DMOG能够降低缺血清所引起的AIF的从胞浆向细胞核的转位,说明DMOG能够降低caspase依赖的和非依赖的凋亡通路而发挥保护作用。缺血清能够明显降低MSCs的Akt和ERK的磷酸化水平,DMOG能够逆转Akt的磷酸化表达水平而不能逆转ERK的活化,提示PI3K/Akt通路而不是MEK/ERK通路可能在DMOG的保护中发挥一定的作用。应用PI3K/Akt通路的特异性阻断剂wortamannin后,发现DMOG逆转的Akt磷酸化水平作用消失,相应地,DMOG对MSCs凋亡和死亡的保护作用(分别由cleaved capase-3和台盼蓝染色检测)也消失了,这从反面也证实了PI3K/Akt通路在DMOG保护中的作用。实验结论:1、DMOG能够降低MSCs内HIF-1α的降解和上调下游Glut-1的表达,在作用浓度下对MSCs没有明显的毒性作用;2、DMOG能够降低缺血清状况下MSCs的凋亡和死亡;3、caspase-3依赖的和非依赖的线粒体通路可能在DMOG对MSCs保护中发挥了一定的作用;4、PI3K/Akt通路在DMOG对MSCs的保护中发挥了重要的作用。更多还原

【Abstract】 Background:Despite great strides in medical strategies aimed at post-infarct remodeling,the development of aggressive reperfusion strategies such as interventional diagnosis and therapy,as well as common drug therapy,heart failure persists as an emerging public health concern in the developing world and remains the leading cause of death and hospitalization in industrialized countries.In recent years,cell-based therapy has emerged as a promising therapeutic approach for restoration of heart function after myocardial infarction.Circumventing ethical and immune rejection issues,autologous mesenchymal stem cells(MSCs) transplantation is an ideal option for clinical cell therapy use.To date,it has been demonstrated that MSCs are capable of ameliorating the cardiac function after myocardial infarction.However,the majority of transplanted cells usually die a few days after transplantation into the harsh host environment.It was reported that over 90%of MSCs died in the first 24 h and only 0.44%survived 4 d after transplantation to ischemic heart.Poor cell viability after transplantation into infarcted myocardium obviously restricts the efficacy of this attractive therapy.Therefore, strategies to enhance MSCs survival are important and urgently needed.Under hypoxic conditions,prolyl hydroxylase activity decreases because they require oxygen as a co-factor and the HIF-1αprotein is stabilized.HIF-1αstabilization is followed by activation of the transcription of numerous target genes such as vascular endothelial growth factor(VEGF),erythropoietin(EPO),and glucose transporter 1 (Glut-1) that are involved in angiogenesis,erythropoiesis,energy metabolism and cell survival.It has been demonstrated that hydroxylases play an important role in heart diseases.Inhibition of prolyl hydroxylase activity or expression can be effected using competitive inhibitors such as dimethyloxalylglycine(DMOG) or siRNA leading to the stabilization of HIF and other beneficial effects.In ischemia-reperfusion(I/R) injury and acute myocardial infarction animal models,inhibition of prolyl hydroxylases has been observed to prevent deleterious remodeling and to attenuate the acute inflammatory response.Additionally,hydroxylase inhibition followed by HIF-1αup-regulation participates in the protection of cell survival and regulation of apoptosis which have been shown in cancer cells and neurons under hypoxic condition,serum deprivation or tropic factor deprivation.The precise role of HIF-1αin regulation cell death and apoptosis remains controversial because of the concern that HIF-1αmay promote cell death.The role of prolyl hydroxylase inhibition by DMOG in regulation of cell survival has not been investigated in MSCs.The purpose of this study is to evaluate the effect of DMOG on the apoptosis and cell death induced by serum deprivation in MSCs and to delineate the underlying signaling mechanisms.Objectives:The objective of this study is to evaluate the effect DMOG on MSCs survival against serum deprivation and to explore the possible mechanisms.Methods:The typical apoptotic model of serum deprivation was used in this study to mimic the microenvironment of ischemic heart diseases in vivo.Three groups,Control group,serum deprivation group(SD) and serum deprivation with DMOG group (SD+DMOG),were divided.According to the concentration of DMOG application, SD+DMOG group was sub-divided into 100μM,500μM and 1000μM groups. Western-blot was applied to detect HIF-1αand its downstream Glut-1 expression of MSCs.Trypan blue was used to detect the cell toxicity in the normal culture condition and MSCs death under serum deprivation.Apoptosis of MSCs in different groups was observed by morphological changes,caspase-3 activation.Morphological anlysis was detected by phase-contrast microscope and Hoechst staining which was checked under fluorescent microscope to detect the nuclear condensation and fragmentation.Apoptotic rate was calculated by cleaved caspase-3 staining and western-blot of cleaved caspase-3 was used to confirm staining results.To evaluate the mechanisms beside HIF-1αpathway,mitochondrial and survival pathways were detected.To study involvement of the mitochondrial pathway in the mechanism,western-blot was used to detect cytochrome C release and apoptosis inducing factor(AIF) translocation to evaluate caspase dependent or caspase independent pathway,respectively.Akt and ERK phosophorylation level were detected by western-blot to evaluate the possible effect of PI3K/Akt and MEK/ERK pathway.Wortmannin,a specific inhibitor of PI3K,was used to block PI3K/Akt pathway and then Akt activation,cleaved caspase-3,and trypan-blue staining were re-evaluated to confirm the involvement of PI3K/Akt pathway in the protective effect of DMOG on MSCs.Results:DMOG remarkably increased MSCs HIF-1αstablizaion and Glut-1 expression without obvious toxicity.DMOG ameliorated the morphological changes by serum deprivation and decreased nuclear condensation detected by Hoechst staining.DMOG significantly reduced the apoptotic rate calculated by cleaved caspase-3 staining in a dose dependent manner which was confirmed by western-blot analysis of cleaved caspase-3 activation.What’s more,DMOG prevented caspase-3 activation in a time course from 6h to 24h.Similarly,DMOG decreased MSCs death in a dose-dependent manner and in a time course from 12h to 72h.Serum deprivation induced cytochrome C release from mitochondria into cytosol and AIF translocation from cytosol into nucleus. DMOG decreased both of of them indicating both caspase dependent and independent apoptotic pathway were possibly involved in the protective effect.Furthermore,serum deprivation significantly decreased Akt and ERK activation compared with control group,while DMOG reversed Akt but not ERK phosphorylation,indicating PI3K/Akt but not MEK/ERK pathway maybe involved in the mechanism.To confirm this, wortmannin,a specific PI3K inhibitor,was used to block PI3K/Akt pathway and Akt activation was inhibited.Meanwhile,wortmannin abrogated the beneficial effect of DMOG on MSCs apoptosis and cell death re-evaluated by cleaved caspase-3 and trypan-blue staining,respectively.Conclusions:The prolyl hydroxylase inhibitor DMOG inhibited apoptosis and cell death induced by serum deprivation in MSCs concurrent with HIF-1αstabilization, mitochondrial protection,and activation of the PI3K/Akt pathway.更多还原