【作者】 刘乐；

【导师】 高国栋； 贾栋；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 博士

【摘要】 【背景】脑血管病是当前严重危害人类生命和健康的常见病之一,其发病之快、恢复之慢、死亡之多、致残之重,给人们带来巨大的身心痛苦和经济损失。脑血管病一般分为出血性脑血管病和缺血性脑血管病两大类,缺血性脑血管病约占所有脑血管病的75%。缺血-再灌注损伤,是指组织缺血一段时间,当血流重新恢复后,组织的损伤程度较缺血时进一步加重、器官功能进一步恶化的综合征,如何减轻再灌注损伤成为提高缺血性脑卒中疗效的关键之一。大量研究结果表明脑缺血再灌注后脑组织自由基生成过多是造成再灌注损伤的主要原因之一,自由基生成过多是级联反应导致细胞损伤的重要病理环节,能导致继发性神经元损伤。虽然说自由基理论已得到广泛的认可,但是对以“超氧化物歧化酶(SOD)”为首的许多抗氧化剂进行的的研究结果卻并不那么令人振奋,SOD作为中等大小的蛋白质,必须进入细胞内甚至是线粒体内才能起到抗氧化损伤的作用,当如此大的蛋白质做为药物使用时,如何使这样大的分子穿过细胞膜和线粒体膜起作用,是首先要解决的难题。本课题通过构建TAT-SOD融合蛋白,并对其在脑缺血再灌注损伤中对抗神经细胞凋亡的能力进行研究,探讨TAT介导肽类或蛋白质类生物大分子药物通过生物屏障的能力以及TAT-SOD的脑保护作用,为临床治疗ROS引发的中枢神经系统疾病提供广阔的前景。【目的】1、构建、表达及纯化TAT-SOD融合蛋白;2、研究目的蛋白TAT-SOD在体外培养的细胞中的跨膜功能;3、观察TAT-SOD在脑缺血再灌注大鼠模型中对凋亡神经细胞的影响,探讨其病理生理机制及临床应用前景。【方法】1、采用PCR及基因重组技术建立TAT-SOD融合蛋白,而后用SDS-PAGE和Western blot对目的蛋白进行检测;2、采用细胞培养技术及体外实验,通过Western blot分析TAT-SOD的跨膜作用;3、应用线栓法建立大鼠局灶性脑缺血再灌注模型,测定对照组及干预组的MDA含量及SOD活性;4、采用免疫组织化学及流式细胞术探讨TAT-SOD融合蛋白对脑缺血再灌注后凋亡神经细胞的影响。【结果】1、成功构建了含有TAT-SOD融合基因的质粒,12% SDS-PAGE及Western blot分析均提示在相对分子质量25000处出现目标表达条带,蛋白以天然的、可溶性的形式存在;2、转导入体外培养的星状胶质细胞内蛋白的量随着TAT-SOD与细胞的孵育时间或蛋白浓度的增加而增加,表现出时间依赖性和剂量依赖性的特点;3、采用颈内动脉线栓法成功制备局灶性脑缺血再灌注大鼠模型,并选择缺血120min恢复再灌注作为时间点,经行为学评分,获得较稳定的神经功能缺失症状;4、对脑缺血-再灌注后脑组织MDA含量及SOD活性进行测定,结果表明MDA含量于再灌注6h开始升高,24h达高峰,之后逐渐下降,SOD活性变化与之相反,TAT-SOD处理组MDA、SOD的动态变化趋势与对照组一致,但各时点值均较低;5、caspase3染色分析提示脑缺血再灌注48小时在假手术组可见少数caspase3阳性细胞,生理盐水对照组及SOD处理组表达明显增加,经TAT-SOD干预后,caspase3表达降低;6、流式细胞术提示脑缺血再灌注后缺血组细胞凋亡率明显升高,SOD处理组与之无显著性差异,TAT-SOD处理组的细胞凋亡率低于缺血组。【结论】1、用TA克隆法构建了含有TAT-SOD融合基因的质粒,通过在大肠杆菌中进行表达,获得了具有SOD酶活力的融合蛋白;2、TAT能以天然活性形式高效介导SOD进入体外培养的星状胶质细胞,并表现出剂量依赖性、时间依赖性的特点;3、颈内动脉线栓法制备局灶性脑缺血再灌注大鼠模型是成熟的动物模型,我们成功建立动物模型,经行为学评分,获得较稳定的神经功能缺失症状,与文献报道(White.BC等)一致;4、脑缺血-再灌注后脑组织MDA含量呈逐渐上升趋势,SOD的活性变化与之相反,经TAT-SOD干预后MDA、SOD的动态变化趋势与对照组一致,但各时点值均较低,提示TAT-SOD有降低自由基水平的作用;5、在假手术组可见少数caspase3阳性细胞,可能是正常生理性死亡或手术剌激所致;缺血120min caspase3表达增加,经TAT-SOD干预,表达降低,SOD处理组无明显变化,提示TAT-SOD保持了蛋白的生物学活性,减少神经细胞的凋亡;6、采用FCM法对凋亡神经细胞进行了定量分析,生理盐水对照组细胞凋亡率明显升高,SOD处理组与之无显著性差异,TAT-SOD处理组的细胞凋亡率低于二者,表明TAT-SOD有效地清除自由基,抑制细胞凋亡,减轻脑组织损伤。更多还原

【Abstract】 【Background】The reactive-oxygen species are inevitably formed as byproducts of various, normal cellular processes involving interactions with oxygen. These reactive- oxygen species damage macromolecules in the cells, and therefore, sometimes make significant contributions to the several pathological processes of ischemia and reperfusion injury.Indeed, numerous studies have demonstrated that antioxidant enzymes can provide substantial protection against oxidative stresses. Also, recombinant enzymes such as SOD have been used to protect against oxidative stresses. These enzymes were modified to obtain long circulating half-lives and to target specificity by conjugation with polyethylene glycol, albumin, antibody, or encapsidation with liposome. Although a variety of modified recombinant enzymes are available to protect against oxidative stresses, one of the major limitations in using these enzymes is the lack of their efficient transduction ability into cells, thus resulting in an inability to detoxify intracellular ROS.In the present study, we describe the transduction of fulllength TAT-SOD fusion protein into astrocytes and whether this transduced TAT-SOD has a protective effect against oxidative stress in the cells.【Objectives】1. Expression and purification of TAT-SOD; 2. Transduction of TAT-SOD into astrocytes; 3. Induction of transient forebrain ischemia, discover the effect of transduced TAT-SOD on neuronal viability after ischemic insult.【Methods】1. Product the TAT-SOD fusion proteins, which were confirmed by SDS-PAGE and western blot analysis.2. Analyz the effect of transduced Tat-SOD on the viability of astrocytes.3.Found models of cerebral ischemia and reperfusion, and calculate the MDA and SOD of the models.4. Analyz the effect of transduced Tat-SOD on neuronal viability afterischemic insult.【Results】1.TAT-SOD and SOD proteins were highly expressed and soluble in E. coli. The identities of the overexpressed proteins were confirmed by SDS-PAGE and Western blot analysis with monoclonal antibody.2. To evaluate the transducing ability of TAT-SOD, we analyzed the kinetics of its incorporation, by adding it to the astrocyte culture medium and measuring the level of the transduced protein by Western blotting.The activity of transduced SOD increased in a time-and dose-dependent manner.3. We found models of cerebral ischemia and reperfusion, which were evaluated successfully in behavior.4. Calculate the MDA and SOD after cerebral ischemia and reperfusion, MDA increased and SOD decreased respectively.5. We tested the effect of transduced Tat-SOD on neuronal cell viability after administering transient forebrain ischemia by Caspase3 and FCM. The observations show that Tat-SOD passes efficiently across the blood-brain barrier and protect cells against cell death. 【Conclusions】1. The SOD gene was fused with a gene fragment encoding the TAT protein transduction domain of HIV-1 in a bacterial expression vector to produce genetic inframe TAT-SOD fusion proteins.2. We found that TAT-SOD fusion protein is efficiently transduced into astrocytes and that when done so it retains its enzymatic and biologic activities in vitro. The activity of transduced SOD increased in a time-and dose-dependent manner.3. The models of cerebral ischemia and reperfusion were evaluated successfully in behavior. 4.TAT-SOD fusion protein is efficiently taken up into neuronal cells,which resulted in MDA increasing and SOD decreasing slowly respectively by eliminate ROS.5. TAT-SOD prevented neuronal cell death in the hippocampus in response to transient forebrain ischemia. These results suggest that Tat-SOD provides a strategy for therapeutic delivery in various human diseases.更多还原