靶向室管膜区胶原的脑源性神经营养因子治疗大鼠脑缺血卒中的实验研究
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摘要
研究背景和目的
脑血管病(cerebral vascular diseases, CVD)是危害人类健康及生命的三大疾病之一,而缺血性脑血管病(ischemic cerebralvascular disease, ICD)占其中大部分。我国脑血管病的患病率高,且有缓慢上升的趋势,每年新发病例超过150万例。临床死亡率高,后遗症多,迫切需要切实有效的治疗方法。
目前,治疗ICD除了发病急性期3-6小时内的溶栓治疗外,尚没有更为有效的治疗措施。但由于溶栓治疗的严格时间窗限制,临床中符合治疗条件的病例即使在欧美发达国家占总病例的比例也非常低,因此,临床上大部分的脑缺血治疗方案为对症保守治疗,效果难以令人满意。
近年,随着营养因子治疗在相关领域的进展,多种营养因子被用于脑缺血疾病临床前研究,并且取得令人振奋的动物实验结果。越来越多的研究者积极推动这一研究成果向临床应用转化。脑源性神经营养因子(brain derived neuro trophic factor, BDNF)是在脑内合成的一种蛋白质,它广泛分布于中枢神经系统内,在中枢神经系统发育过程中,对神经元的存活、分化、生长发育起重要作用,并能防止神经元受损伤死亡、改善神经元的病理状态、促进受损伤神经元再生及分化等生物效应。BDNF在脑缺血、脊髓损伤、周围神经损伤、帕金森病等的研究中已经取得一定进展。
因神经营养因子半衰期短,在局部维持和浓聚困难,治疗神经损伤时,需要持续静脉注射、反复多次给药,或者应用人工合成的材料缓释系统,增加感染及损伤等风险。
CBD (collagen-bingding domain)为多肽结构,是研究胶原酶作用位点时发现的胶原结合区。应用PCR技术,将CBD-BDNF基因扩增,插入pET-28a载体,后转染E.coli,使E.coli表达目标蛋白,在神经营养因子结构基础上以一个CBD多肽结构修饰,行成CBD-BDNF。可使BDNF铆定于胶原表面,从而形成缓释系统。实验证明有CBD多肽的神经营养因子对神经损伤的修复作用明显好于普通神经营养因子。
在大鼠的室管膜区,分布着大量Ⅰ型胶原,同时室管膜下区(subventricular zone, SVZ)为内源性神经祖细胞增殖的主要场所。因此,本课题的目的是研究靶向室管膜区胶原的BDNF脑内移植治疗缺血性脑卒中的效果,探讨CBD-BDNF治疗缺血性脑卒中的是否可以结合与室管膜区,在时间上形成缓释系统,作用时间更为长久,在空间上,更加直接的刺激室管膜下区的内源性神经祖细胞增殖,从而促进脑缺血损伤的恢复,为营养因子治疗缺血性脑卒中的临床应用提供实验依据。
研究内容和方法
拟建立大鼠大脑中动脉梗阻(middle cerebral atery occlusion, MCAO)脑缺血模型,以动物模型,研究胶原靶向的脑源性神经营养因子对脑缺血再灌注损伤的作用,为临床上选择高效的神经营养因子提供实验依据。课题研究共分为两部分。第一部分：以永久线栓法制作大鼠急性脑缺血模型,随机分为磷酸盐缓冲液组,BDNF治疗组。在侧脑室内分别注射NAT-BDNF10ul (0.5nmol), CBD-BDNF10ul (0.5nmol),磷酸盐缓冲液10ul。在注射后3h,12h,通过western评价营养因子是否能在局部形成缓释系统。治疗后2周,三组分别行神经功能学评分、免疫组化染色、Micro-PET, SPECT扫描,评价CBD-BDNF治疗大鼠脑缺血损伤的有效性及可能的作用机制。第二部分：评价micro-Single Photon Emission Computed Tomography/Computed Tomography (SPECT/CT)对大鼠活体状态下超急性期脑缺血的诊断价值。应用99mTc-双半胱乙酯(ECD)脑灌注断层显像技术,选取24只健康雄性成年SD大鼠,以线栓法制作大鼠急性脑缺血模型,术后6h内每隔1h分别行SPECT/CT检查、2,3,5三苯基氯化四氮唑(TTC)染色及HE染色。对组内不同时间点间梗死体积的比较采用单因素ANOVA,同一时间点TTC及SPECT/CT结果比较采用独立样本t检验。
研究结果
1.通过western blot及免疫组化证实,大鼠脑内室管膜分布丰富Ⅰ型胶原。
2.以缺血损伤侧的脑室室管膜胶原基质为靶点,使CBD-BDNF在脑缺血损伤局部维持和浓聚,从而形成一个缓释系统,在注射后12h,脑室内已基本无NAT-BDNF,但仍可发现CBD-BDNF在局部浓聚。
3.CBD结合区明显增强BDNF对损伤的修复效果。CBD-BDNF治疗组神经功能恢复好于NAT-BDNF组及磷酸盐缓冲液组,在室管膜下区域细胞增殖活跃。CBD-BDNF治疗组梗塞周边区的神经元存活数目及血管密度明显多于磷酸盐缓冲液组,micro-PET结果显示梗塞侧有代谢活性脑组织增多。SPECT/CT灌注显像显示灌注缺失区域明显减少。
4.脑源性神经营养因子能改善脑损伤大鼠的功能；上调Bcl-2蛋白表达,下调Bax蛋白表达,减少神经细胞凋亡,可能是其主要机制之一。
5. SPECT所示缺血体积与TTC染色梗死区体积比较无统计学差异(P>0.05)。SPECT显示的低血流灌注区域,表现为放射性稀疏区域,与TTC染色粉红色区域相对应。缺血3h及以后梗塞面积基本趋于恒定,3h、4h、5h、6h各时间点梗塞面积比较无统计学意义(P>0.05)。HE染色表现：缺血1h、2h血管间隙增宽,神经细胞水肿；3h、4h、5h、6h,神经核固缩,血管周围大量空泡状改变。
结论
1. CBD-BDNF脑室内注射可以显著改善脑缺血大鼠神经功能的恢复,促进室管膜下区内源性神经祖细胞增殖,保护神经元,促进血管新生,抑制细胞凋亡。
2. Micro-SPECT/CT可在活体状态下快速、准确、无创地评价脑缺血动物模型的脑部血流动力学改变,对超急性期脑梗死的评估及指导治疗决策提供有力的依据。
Backround and Objective
Cerebral ischemic stroke is one of the most common diseases affecting human health. It has a high mortality rate and many clinically difficult sequelae such as hemiplegia, numbness, dysphasia and cognitive deficits.
Neurotrophic factors are important for the treatment of cerebral ischemia. One of the most widely distributed neurotrophic factors in the central nervous system is BDNF, which may promote neural regeneration and angiogenesis, and decrease apoptosis and infarct volume. Although there is an upregulation of BDNF expression after cerebral traumatic events, the amount of endogenous BDNF expressed is not sufficient to promote recovery. Therefore, exogenous supplementation of BDNF has high potential for the clinical treatment of stroke.
Because BDNF is poorly transported through the blood-brain barrier (BBB), the efficacy of intravenous administration is not clear and it is difficult to control the amount of BDNF delivered. Intraventricular injection is a more effective method for administration of BNDF. Owning to its short half-life and rapid diffusion to cerebrospinal fluid, multi-injections of BDNF or use of an osmotic minipump are currently necessary to maintain local concentrations in the brain, leading to increased wound complications and the risk of infection. Hence, development of a sustained single dose BDNF delivery system is crucial.
We have previously demonstrated that BDNF fused with a collagen-binding domain (CBD) specifically binds to collagen. We also found that collagen is abundant in the ventricular ependyma of the brain. Therefore, the collagen of the ventricular ependyma of brain may provide a binding target for CBD-BDNF. Moreover, the subventricular zone (SVZ) is the main region for the generation of new neurons. We proposed that CBD-BDNF would act as an ingenious natural slow release system that would directly stimulate the cell proliferation of the SVZ and exert a therapeutic effect for a longer time. In this study, our collagen-targeting therapeutic strategy was tested in the rat middle cerebral artery occlusion model (MCAO) and the therapeutic effects were estimated by molecular imaging technology, immunohistochemistry and ethology score.
Methods
Based on rats MCAO model, The study includes two parts:
1. Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery of adult rats. At8h after the permanent MCAO surgery, the infarct size become stable. Animals were randomly divided into three groups: NAT-BDNF-grafted, CBD-BDNF-grafted, and medium-grafted groups. Each rat received10ul (0.5nmol) NAT-BDNF,10ul (0.5nmol) CBD-BDNF or10ul PBSinjection slowly for1min into the right lateral ventricle by a stereotactic frame respectively.24rats (n=6, each group of each time point) were executed by cervical dislocation at3h or12h after injection of NAT-BDNF or CBD-BDNF. The infarcted hemisphere was removed and frozen immediately in liquid nitrogen. Then proteins were extracted for western blotting analysis. The Behavioral tests, immunohistochemistry and Micro-PET,SPECT were performed in each group at2weeks after MCAO.
2. A stable and permanent acute cerebral ischemia model with unilateral middle cerebral occlusion was established in SD rats and evaluated by micro-SPECT/CT, TTC staining and HE staining within6h of stroke onset once an hour. An independent sample t-test was used to determine the statistical differences between SPECT imaging and TTC staining. A one-way ANOVA was used to analyze multiple comparison procedures.
Results
1. The distribution of collagen in the ventricular ependyma and choroid plexus of the brain was tested by immunohistochemistry and western blot analysis.
2. At both3h and12h after injection, there was significantly more CBD-BDNF remaining in the infarcted hemisphere compared to NAT-BDNF
3. Significant functional improvement were observed in the CBD-BDNF treatment group starting at one week after MCAO. The modified neurological severity score of CBD-BDNF group seem to be lower than the control group which demonstrate the better neurological recovery. Administration of CBD-BDNF increased the number of Ki-67-positive cells in the ischemic ipsilateral SVZ compared with the medium control.There are more microvessels in the cortical peri-infarct zones of BDNF group. The number of NeuN immunoreactive mature neurons was also higher in the BDNF group compared to the control groupat the2weeks postischemia. SPECT also showed a significant reduction of the radial rarefaction and defect area in the CBD-BDNF group.18FDG-PET examination displayed a significant reduction of the radial rarefaction and defect area in the CBD-BDNF group.
4. In the CBD-BDNF treated group, the number of apoptotic neural cells and the expression of Bax were lower, while the expression of Bcl-2significantly increased
5. The infarction zone obtained from SPECT were concordant with the infarction volume from TTC staining for (P>0.05). There was a positive correlation between SPECT low volume infusion areas and the pink areas by TTC staining. The infarct size tends to be constant3h after the occlusion. At the time points of3h、4h、5h、6h there was no significant difference in infarct size.
Conclusion
CBD-BDNF bound to the collagen of the ventricular ependyma and exerted therapeutic effects more efficiently than NAT-BDNF. In the infarcted hemisphere, CBD-BDNF remained at a higher concentration than NAT-BDNF and stimulated cell proliferation in the SVZ. In the MCAO model, CBD-BDNF promoted cell proliferation, improved perfusion, reduced cell loss, decreased apoptosis, and improved functional recovery in the rat, suggesting that CBD-BDNF may provide an exciting potential treatment for stroke.
SPECT as a rapid, accurate, non-invasive method can evaluate the brain haemodynamics of cerebral ischemia animal model in living state, and may have important clinical value for the ultra-acute cerebral infarction for evaluation and decision making.
脑血管病(cerebral vascular diseases, CVD)是危害人类健康及生命的三大疾病之一,而缺血性脑血管病(ischemic cerebralvascular disease, ICD)占其中大部分。我国脑血管病的患病率高,且有缓慢上升的趋势,每年新发病例超过150万例。临床死亡率高,后遗症多,迫切需要切实有效的治疗方法。
目前,治疗ICD除了发病急性期3-6小时内的溶栓治疗外,尚没有更为有效的治疗措施。但由于溶栓治疗的严格时间窗限制,临床中符合治疗条件的病例即使在欧美发达国家占总病例的比例也非常低,因此,临床上大部分的脑缺血治疗方案为对症保守治疗,效果难以令人满意。
近年,随着营养因子治疗在相关领域的进展,多种营养因子被用于脑缺血疾病临床前研究,并且取得令人振奋的动物实验结果。越来越多的研究者积极推动这一研究成果向临床应用转化。脑源性神经营养因子(brain derived neuro trophic factor, BDNF)是在脑内合成的一种蛋白质,它广泛分布于中枢神经系统内,在中枢神经系统发育过程中,对神经元的存活、分化、生长发育起重要作用,并能防止神经元受损伤死亡、改善神经元的病理状态、促进受损伤神经元再生及分化等生物效应。BDNF在脑缺血、脊髓损伤、周围神经损伤、帕金森病等的研究中已经取得一定进展。
因神经营养因子半衰期短,在局部维持和浓聚困难,治疗神经损伤时,需要持续静脉注射、反复多次给药,或者应用人工合成的材料缓释系统,增加感染及损伤等风险。
CBD (collagen-bingding domain)为多肽结构,是研究胶原酶作用位点时发现的胶原结合区。应用PCR技术,将CBD-BDNF基因扩增,插入pET-28a载体,后转染E.coli,使E.coli表达目标蛋白,在神经营养因子结构基础上以一个CBD多肽结构修饰,行成CBD-BDNF。可使BDNF铆定于胶原表面,从而形成缓释系统。实验证明有CBD多肽的神经营养因子对神经损伤的修复作用明显好于普通神经营养因子。
在大鼠的室管膜区,分布着大量Ⅰ型胶原,同时室管膜下区(subventricular zone, SVZ)为内源性神经祖细胞增殖的主要场所。因此,本课题的目的是研究靶向室管膜区胶原的BDNF脑内移植治疗缺血性脑卒中的效果,探讨CBD-BDNF治疗缺血性脑卒中的是否可以结合与室管膜区,在时间上形成缓释系统,作用时间更为长久,在空间上,更加直接的刺激室管膜下区的内源性神经祖细胞增殖,从而促进脑缺血损伤的恢复,为营养因子治疗缺血性脑卒中的临床应用提供实验依据。
研究内容和方法
拟建立大鼠大脑中动脉梗阻(middle cerebral atery occlusion, MCAO)脑缺血模型,以动物模型,研究胶原靶向的脑源性神经营养因子对脑缺血再灌注损伤的作用,为临床上选择高效的神经营养因子提供实验依据。课题研究共分为两部分。第一部分：以永久线栓法制作大鼠急性脑缺血模型,随机分为磷酸盐缓冲液组,BDNF治疗组。在侧脑室内分别注射NAT-BDNF10ul (0.5nmol), CBD-BDNF10ul (0.5nmol),磷酸盐缓冲液10ul。在注射后3h,12h,通过western评价营养因子是否能在局部形成缓释系统。治疗后2周,三组分别行神经功能学评分、免疫组化染色、Micro-PET, SPECT扫描,评价CBD-BDNF治疗大鼠脑缺血损伤的有效性及可能的作用机制。第二部分：评价micro-Single Photon Emission Computed Tomography/Computed Tomography (SPECT/CT)对大鼠活体状态下超急性期脑缺血的诊断价值。应用99mTc-双半胱乙酯(ECD)脑灌注断层显像技术,选取24只健康雄性成年SD大鼠,以线栓法制作大鼠急性脑缺血模型,术后6h内每隔1h分别行SPECT/CT检查、2,3,5三苯基氯化四氮唑(TTC)染色及HE染色。对组内不同时间点间梗死体积的比较采用单因素ANOVA,同一时间点TTC及SPECT/CT结果比较采用独立样本t检验。
研究结果
1.通过western blot及免疫组化证实,大鼠脑内室管膜分布丰富Ⅰ型胶原。
2.以缺血损伤侧的脑室室管膜胶原基质为靶点,使CBD-BDNF在脑缺血损伤局部维持和浓聚,从而形成一个缓释系统,在注射后12h,脑室内已基本无NAT-BDNF,但仍可发现CBD-BDNF在局部浓聚。
3.CBD结合区明显增强BDNF对损伤的修复效果。CBD-BDNF治疗组神经功能恢复好于NAT-BDNF组及磷酸盐缓冲液组,在室管膜下区域细胞增殖活跃。CBD-BDNF治疗组梗塞周边区的神经元存活数目及血管密度明显多于磷酸盐缓冲液组,micro-PET结果显示梗塞侧有代谢活性脑组织增多。SPECT/CT灌注显像显示灌注缺失区域明显减少。
4.脑源性神经营养因子能改善脑损伤大鼠的功能；上调Bcl-2蛋白表达,下调Bax蛋白表达,减少神经细胞凋亡,可能是其主要机制之一。
5. SPECT所示缺血体积与TTC染色梗死区体积比较无统计学差异(P>0.05)。SPECT显示的低血流灌注区域,表现为放射性稀疏区域,与TTC染色粉红色区域相对应。缺血3h及以后梗塞面积基本趋于恒定,3h、4h、5h、6h各时间点梗塞面积比较无统计学意义(P>0.05)。HE染色表现：缺血1h、2h血管间隙增宽,神经细胞水肿；3h、4h、5h、6h,神经核固缩,血管周围大量空泡状改变。
结论
1. CBD-BDNF脑室内注射可以显著改善脑缺血大鼠神经功能的恢复,促进室管膜下区内源性神经祖细胞增殖,保护神经元,促进血管新生,抑制细胞凋亡。
2. Micro-SPECT/CT可在活体状态下快速、准确、无创地评价脑缺血动物模型的脑部血流动力学改变,对超急性期脑梗死的评估及指导治疗决策提供有力的依据。
Backround and Objective
Cerebral ischemic stroke is one of the most common diseases affecting human health. It has a high mortality rate and many clinically difficult sequelae such as hemiplegia, numbness, dysphasia and cognitive deficits.
Neurotrophic factors are important for the treatment of cerebral ischemia. One of the most widely distributed neurotrophic factors in the central nervous system is BDNF, which may promote neural regeneration and angiogenesis, and decrease apoptosis and infarct volume. Although there is an upregulation of BDNF expression after cerebral traumatic events, the amount of endogenous BDNF expressed is not sufficient to promote recovery. Therefore, exogenous supplementation of BDNF has high potential for the clinical treatment of stroke.
Because BDNF is poorly transported through the blood-brain barrier (BBB), the efficacy of intravenous administration is not clear and it is difficult to control the amount of BDNF delivered. Intraventricular injection is a more effective method for administration of BNDF. Owning to its short half-life and rapid diffusion to cerebrospinal fluid, multi-injections of BDNF or use of an osmotic minipump are currently necessary to maintain local concentrations in the brain, leading to increased wound complications and the risk of infection. Hence, development of a sustained single dose BDNF delivery system is crucial.
We have previously demonstrated that BDNF fused with a collagen-binding domain (CBD) specifically binds to collagen. We also found that collagen is abundant in the ventricular ependyma of the brain. Therefore, the collagen of the ventricular ependyma of brain may provide a binding target for CBD-BDNF. Moreover, the subventricular zone (SVZ) is the main region for the generation of new neurons. We proposed that CBD-BDNF would act as an ingenious natural slow release system that would directly stimulate the cell proliferation of the SVZ and exert a therapeutic effect for a longer time. In this study, our collagen-targeting therapeutic strategy was tested in the rat middle cerebral artery occlusion model (MCAO) and the therapeutic effects were estimated by molecular imaging technology, immunohistochemistry and ethology score.
Methods
Based on rats MCAO model, The study includes two parts:
1. Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery of adult rats. At8h after the permanent MCAO surgery, the infarct size become stable. Animals were randomly divided into three groups: NAT-BDNF-grafted, CBD-BDNF-grafted, and medium-grafted groups. Each rat received10ul (0.5nmol) NAT-BDNF,10ul (0.5nmol) CBD-BDNF or10ul PBSinjection slowly for1min into the right lateral ventricle by a stereotactic frame respectively.24rats (n=6, each group of each time point) were executed by cervical dislocation at3h or12h after injection of NAT-BDNF or CBD-BDNF. The infarcted hemisphere was removed and frozen immediately in liquid nitrogen. Then proteins were extracted for western blotting analysis. The Behavioral tests, immunohistochemistry and Micro-PET,SPECT were performed in each group at2weeks after MCAO.
2. A stable and permanent acute cerebral ischemia model with unilateral middle cerebral occlusion was established in SD rats and evaluated by micro-SPECT/CT, TTC staining and HE staining within6h of stroke onset once an hour. An independent sample t-test was used to determine the statistical differences between SPECT imaging and TTC staining. A one-way ANOVA was used to analyze multiple comparison procedures.
Results
1. The distribution of collagen in the ventricular ependyma and choroid plexus of the brain was tested by immunohistochemistry and western blot analysis.
2. At both3h and12h after injection, there was significantly more CBD-BDNF remaining in the infarcted hemisphere compared to NAT-BDNF
3. Significant functional improvement were observed in the CBD-BDNF treatment group starting at one week after MCAO. The modified neurological severity score of CBD-BDNF group seem to be lower than the control group which demonstrate the better neurological recovery. Administration of CBD-BDNF increased the number of Ki-67-positive cells in the ischemic ipsilateral SVZ compared with the medium control.There are more microvessels in the cortical peri-infarct zones of BDNF group. The number of NeuN immunoreactive mature neurons was also higher in the BDNF group compared to the control groupat the2weeks postischemia. SPECT also showed a significant reduction of the radial rarefaction and defect area in the CBD-BDNF group.18FDG-PET examination displayed a significant reduction of the radial rarefaction and defect area in the CBD-BDNF group.
4. In the CBD-BDNF treated group, the number of apoptotic neural cells and the expression of Bax were lower, while the expression of Bcl-2significantly increased
5. The infarction zone obtained from SPECT were concordant with the infarction volume from TTC staining for (P>0.05). There was a positive correlation between SPECT low volume infusion areas and the pink areas by TTC staining. The infarct size tends to be constant3h after the occlusion. At the time points of3h、4h、5h、6h there was no significant difference in infarct size.
Conclusion
CBD-BDNF bound to the collagen of the ventricular ependyma and exerted therapeutic effects more efficiently than NAT-BDNF. In the infarcted hemisphere, CBD-BDNF remained at a higher concentration than NAT-BDNF and stimulated cell proliferation in the SVZ. In the MCAO model, CBD-BDNF promoted cell proliferation, improved perfusion, reduced cell loss, decreased apoptosis, and improved functional recovery in the rat, suggesting that CBD-BDNF may provide an exciting potential treatment for stroke.
SPECT as a rapid, accurate, non-invasive method can evaluate the brain haemodynamics of cerebral ischemia animal model in living state, and may have important clinical value for the ultra-acute cerebral infarction for evaluation and decision making.
引文
1. Beal CC. Gender and stroke symptoms:a review of the current literature. J Neurosci Nurs 2010 2010-04-01;42(2):80-87.
2. Rymer MM, Summers D. Ischemic stroke:prevention of complications and secondary prevention. Mo Med 2010 2010-11-01;107(6):396-400.
3. Gialanella B, Ferlucci C. Functional outcome after stroke in patients with aphasia and neglect:assessment by the motor and cognitive functional independence measure instrument. Cerebrovasc Dis 2010 2010-01-20;30(5):440-447.
4. Abe K. Therapeutic potential of neurotrophic factors and neural stem cells against ischemic brain injury. J Cereb Blood Flow Metab 2000 2000-10-01;20(10):1393-1408.
5. Schabitz WR, Steigleder T, Cooper-Kuhn CM, Schwab S, Sommer C, Schneider A, et al. Intravenous brain-derived neurotrophic factor enhances poststroke sensorimotor recovery and stimulates neurogenesis. Stroke 2007 2007-07-01;38(7):2165-2172.
6. Kurozumi K, Nakamura K, Tamiya T, Kawano Y, Kobune M, Hirai S, et al. BDNF gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebral artery occlusion model. Mol Ther 2004 2004-02-01;9(2):189-197.
7. Muller HD, Neder A, Sommer C, Schabitz WR. Different postischemic protein expression of the GABA(A) receptor alpha2 subunit and the plasticity-associated protein MAP IB after treatment with BDNF versus G-CSF in the rat brain. Restor Neurol Neurosci 2009 2009-01-20;27(1):27-39.
8. Li B, Piao CS, Liu XY, Guo WP, Xue YQ, Duan WM, et al. Brain self-protection:the role of endogenous neural progenitor cells in adult brain after cerebral cortical ischemia. Brain Res 2010 2010-04-23;1327:91-102.
9. Zhang Y, Pardridge WM. Neuroprotection in transient focal brain ischemia after delayed intravenous administration of brain-derived neurotrophic factor conjugated to a blood-brain barrier drug targeting system. Stroke 2001 2001-06-01;32(6):1378-1384.
10. Yanamoto H, Nagata I, Sakata M, Zhang Z, Tohnai N, Sakai H, et al. Infarct tolerance induced by intra-cerebral infusion of recombinant brain-derived neurotrophic factor. Brain Res 2000 2000-03-24;859(2):240-248.
11. Schabitz WR, Schwab S, Spranger M, Hacke W. Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats. J Cereb Blood Flow Metab 1997 1997-05-01;17(5):500-506.
12. Han Q, Sun W, Lin H, Zhao W, Gao Y, Zhao Y, et al. Linear ordered collagen scaffolds loaded with collagen-binding brain-derived neurotrophic factor improve the recovery of spinal cord injury in rats. Tissue Eng Part A 2009 2009-10-01;15(10):2927-2935.
13. Mercier F, Kitasako JT, Hatton GI. Anatomy of the brain neurogenic zones revisited: fractones and the fibroblast/macrophage network. J Comp Neurol 2002 2002-09-16;451(2):170-188.
14. Kim JT, Park MS, Choi KH, et al. The CBV-ASPECT Score as a predictor of fatal stroke in a hyperacute state. Eur Neurol.2010;63(6):357-63.
1.吴兆苏,姚崇华,赵冬.我国人群脑卒中发病率、死亡率的流行病学研究.中华流行病学杂志.2003；24：236-239。
2.刘承基.脑血管外科学.南京：江苏科学技术出版社;
3. Gialanella B, Ferlucci C. Functional outcome after stroke in patients with aphasia and neglect:assessment by the motor and cognitive functional independence measure instrument. Cerebrovasc Dis 2010 2010-01-20;30(5):440-447.
4. Miiller HD, Hanumanthiah KM, Diederich K, et al. Brain-derived neurotrophic factor but not forced arm use improves long-term outcome after photothrombotic stroke and transiently upregulates binding densities of excitatory glutamate receptors in the rat brain. Stroke.2008, 39(3):1012-1021. Epub 2008 Jan 31.
5.杨小锋,虞军,刘伟国,等。脑源性神经营养因子和神经干细胞联合移植治疗大鼠重度颅脑损伤的研究.浙江医学,2005,27：1-3.
6. Kermani P, Hempstead B. Brain-derived neurotrophic factor:a newly described mediator of angiogenesis. Trends Cardiovasc Med.2007,17(4):140-143.
7.黄东煜,张志坚,陈柏龄,等。慢病毒载体介导Bdnf基因修饰的骨髓间质干细胞移植治疗脑梗死。生物工程学报,2008；24(7)：1174-1179.
8. Li B, Piao CS, Liu XY, Guo WP, Xue YQ, Duan WM, et al. Brain self-protection:the role of endogenous neural progenitor cells in adult brain after cerebral cortical ischemia. Brain Res 2010 2010-04-23;1327:91-102.
9. Zhang Y, Pardridge WM. Neuroprotection in transient focal brain ischemia after delayed intravenous administration of brain-derived neurotrophic factor conjugated to a blood-brain barrier drug targeting system. Stroke 20012001-06-01;32(6):1378-1384.
10. Yanamoto H, Nagata I, Sakata M, Zhang Z, Tohnai N, Sakai H, et al. Infarct tolerance induced by intra-cerebral infusion of recombinant brain-derived neurotrophic factor. Brain Res 2000 2000-03-24;859(2):240-248.
11. Schabitz WR, Schwab S, Spranger M, Hacke W. Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats. J Cereb Blood Flow Metab 1997 1997-05-01;17(5):500-506.
12. Han Q, Sun W, Lin H, Zhao W, Gao Y, Zhao Y, et al. Linear ordered collagen scaffolds loaded with collagen-binding brain-derived neurotrophic factor improve the recovery of spinal cord injury in rats. Tissue Eng Part A 2009 2009-10-01;15(10):2927-2935.
13.8.Teng H, Zhang ZG, Wang L, et al. Coupling of angiogenesis and neurogenesis in cultured endothelial cells and neural progenitor cells after stroke. J Cereb Blood Flow Metab.2008, 28(4):764-771.
14. Yang YJ, Wang XL, Yu XH, et al. Hyperbaric oxygen induces endogenous neural stem cells to proliferate and differentiate in hypoxic-ischemic brain damage in neonatal rats. Undersea Hyperb Med.2008,35(2):113-129.
15.林百喜,谢明,武衡。大脑中动脉阻塞大鼠脑内脑源性神经营养因子蛋白表达变化。中国现代医学杂志,2008,18：702-705.
16. Pencea V, Bingaman KD, Wiegand SJ, et al. Infusion of brain—derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypOthalamusfJ. J Neurosci,2001,21(17):6706-6717.
17. Kim MW, Bang MS, Han TR,et al. Exercise increased BDNF and trkB in the conrralateral hemisphere of the ischemic rat brain. Brain Res.2005,1052(1):16-21.
18.汪志凌,毛萌,周晖,等。脑源性神经营养因子对缺氧胚脑神经元保护作用的信号传递途径初探。四川大学学报(医学版),2007,38：934-937.
19. Sun C, Hu Y, Chu Z, et al. The effect of brain-derived neurotrophic factor on angiogenesis. J Huazhong Univ Sci Technolog Med Sci.2009,29(2):139-143.
20.孙春艳,胡豫,,吴涛,等。脑源性神经营养因子对血管新生的作用.中华病理学杂志,2006,35：238-239.
21. Leventhal C, Rafii S, Rafii D, Shahar A, Goldman SA. Endothelial trophic support of neuronal production and recruitment from the adult mammalian subependyma. Mol Cell Neurosci 1999; 13:450-464.
22. Fu YK, Chang CJ, Chen KY, et al. Imaging of regional metabolic activity by (18)F-FDG/PET in rats with transient cerebral ischemia. Appl Radiat Isot.2009 Oct;67(10):1743-7.
23.郭拮,王瑞民,张锦明,等。阿尔茨海默病大鼠模型PET在体成像研究。首都医科大学学报,2008,29：12-14.
24.8. Jiang Y, Jahagirdar BN, Reinhardt RL. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature,2002,418(6 893):41
1. Kim JT, Park MS, Choi KH, et al. The CBV-ASPECT Score as a predictor of fatal stroke in a hyperacute state. Eur Neurol. 2010; 63 (6):357-63.
2.李培勇,郭万华等.SPM和ROI方法研究脑内?99Tc?m—ECD的稳定性.中华核医学杂志2001；21(4)：235-236
3. Chen R, Liang F, Ishigami K, et al. Clinical risk factors in regional brain ischemia using single photon emission computed tomography. J Am Geriatr Soc.2010;58(7):1411-2.
4. Kiewert C, Mdzinarishvili A, Hartmann J, et al. Metabolic and transmitter changes in core and penumbra after middle cerebral artery occlusion in mice. Brain Res.2010;1312:101-107.
5. Shimosegawa E, Hatazawa J, Inugami A, et al. Cerebral infarction within six hours of onset:prediction of completed infarction with technetium-99m-HMPAO SPECT. J Nucl Med.1994;35(7):1097-1103.
6. Uyttenboogaart M, De Keyser J, Luijckx GJ. Thrombolysis for acute ischemic stroke. Curr Top Med Chem 2009;9(14):1285-1290.
7. Abels B, Klotz E, Tomandl BF, et al. Perfusion CT in acute ischemic stroke:a qualitative and quantitative comparison of deconvolution and maximum slope approach. AJNR Am J Neuroradiol. 2010;31(9):1690-1698.
8. Nuutinen J, Liu Y, Laakso MP, et al. Perfusion differences on SPECT and PWI in patients with acute ischemic stroke. Neuroradiology. 2009;51(10):687-695.
9. Langheinrich AC, Yeniguen M, Ostendorf A, et al. Evaluation of the middle cerebral artery occlusion techniques in the rat by in-vitro 3-dimensional micro- and nano computed tomography. BMC Neurol. 2010;10:36.
10. Liu Z, Barrett HH, Stevenson GD, et al. Evaluating the protective role of ischaemic preconditioning in rat hearts using a stationary small-animal SPECT imager and 99mTc-glucarate. Nucl Med Commun.2008;29(2):120-128.
[1]Spiegelstein 0, Eudy JD, Finnell RH. Identification of two putative novel folate receptor genes in humans and mouse[J], Gene,2000,258 (1-2):117-25.
[2]Antony AC. Folate receptors[J]. Ann Rev Nutr,1996;16:501-21.
[3]Weitman SD, Frazier KM, Kamen BA. The folate receptor in central nervous system malignancies of childhood. Neurooncol,1994,21:107-112.
[4]Todd A. Patrick, David M. Kranz, Terry A. van Dyke et al. Folate receptors as potential therapeutic targets in choroid plexus tumors of SV40 transgenic mice[J]. Journal of Neuro-Oncology,1997,32:111-123.
[5]Oyesiku NM, Evans CO, et al. Differential expression of folate receptor in pituitary adenomas[J]. Cancer Res,2003,63:4218-4224.
[6]Ladino CA, Chari RV, Bourret LA, et al. Folate—maytansinoids: target—selective drugs of low molecular weight[J]. International journal of cancer,1997,73:859-864.
[7]Aronov o, Horowitz AT, Gabizon A, et al. Folate—targeted PEG as a potential carrier for Carboplatin analogs[J]. Synthesis and in vitro studies, Bioconjugate chemistry,2003,14:563-574.
[8]Reddy JA, Dorton R, Westrick E, et al. Preclinical evaluation of EC145, a folate-vinca alkaloid conjugate[J]. Cancer Res 2007,67:4434-4442.
[9]ZHAOX B, LEE R J, et al. Tumor-slectivetargeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor[J]. Adv Drug Ddiv Rev,2004,56(8):1193-1204.
[10]Lu Y, Low PS, et al. Folate targeting of haptens to cancer cell surfaces mediated immunotherapy of syngenic murine tumors[J]. Cancer Immunol Immunother,2002,51:153-162.
[11]Siegel BA, Dehdashti F, Mutch DG, et al. Evaluation of 111In-DTPA-folate as a receptor-targeted diagnostic agent for ovarian cancer:initial clinical results[J].J Nucl Med 2003,44:700-707.
[13]Kalli KR, Oberg AL, Keeney GL, et al. Folate receptor alpha as a tumor target in epithelial ovarian cancer. Gynecol Oncol 2008,108:619-626.
[14]Yamamoto T, Tsuboi K. Particle radiotherapy for malignant gliomas. Brain Nerve 2009,;61 (7):855-66.
[15]梁兵,肖中鹏,李艺,等.新型靶向纳米材料介导基因治疗胶质瘤的体外研究.重庆医科大学学报.2008,33(1)：14-22.
[16]Fani M, Wang X, Nicolas G, Development of new folate-based PET radiotracers:preclinical evaluation of (68)Ga-DOTA-folate conjugates. Eur J Nucl Med Mol Imaging.2010 Aug 27.
[17]The MICAD Research Team. NIR2-Folate.Molecular Imaging and Contrast Agent Database [Internet].2006 May 19
2. Rymer MM, Summers D. Ischemic stroke:prevention of complications and secondary prevention. Mo Med 2010 2010-11-01;107(6):396-400.
3. Gialanella B, Ferlucci C. Functional outcome after stroke in patients with aphasia and neglect:assessment by the motor and cognitive functional independence measure instrument. Cerebrovasc Dis 2010 2010-01-20;30(5):440-447.
4. Abe K. Therapeutic potential of neurotrophic factors and neural stem cells against ischemic brain injury. J Cereb Blood Flow Metab 2000 2000-10-01;20(10):1393-1408.
5. Schabitz WR, Steigleder T, Cooper-Kuhn CM, Schwab S, Sommer C, Schneider A, et al. Intravenous brain-derived neurotrophic factor enhances poststroke sensorimotor recovery and stimulates neurogenesis. Stroke 2007 2007-07-01;38(7):2165-2172.
6. Kurozumi K, Nakamura K, Tamiya T, Kawano Y, Kobune M, Hirai S, et al. BDNF gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebral artery occlusion model. Mol Ther 2004 2004-02-01;9(2):189-197.
7. Muller HD, Neder A, Sommer C, Schabitz WR. Different postischemic protein expression of the GABA(A) receptor alpha2 subunit and the plasticity-associated protein MAP IB after treatment with BDNF versus G-CSF in the rat brain. Restor Neurol Neurosci 2009 2009-01-20;27(1):27-39.
8. Li B, Piao CS, Liu XY, Guo WP, Xue YQ, Duan WM, et al. Brain self-protection:the role of endogenous neural progenitor cells in adult brain after cerebral cortical ischemia. Brain Res 2010 2010-04-23;1327:91-102.
9. Zhang Y, Pardridge WM. Neuroprotection in transient focal brain ischemia after delayed intravenous administration of brain-derived neurotrophic factor conjugated to a blood-brain barrier drug targeting system. Stroke 2001 2001-06-01;32(6):1378-1384.
10. Yanamoto H, Nagata I, Sakata M, Zhang Z, Tohnai N, Sakai H, et al. Infarct tolerance induced by intra-cerebral infusion of recombinant brain-derived neurotrophic factor. Brain Res 2000 2000-03-24;859(2):240-248.
11. Schabitz WR, Schwab S, Spranger M, Hacke W. Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats. J Cereb Blood Flow Metab 1997 1997-05-01;17(5):500-506.
12. Han Q, Sun W, Lin H, Zhao W, Gao Y, Zhao Y, et al. Linear ordered collagen scaffolds loaded with collagen-binding brain-derived neurotrophic factor improve the recovery of spinal cord injury in rats. Tissue Eng Part A 2009 2009-10-01;15(10):2927-2935.
13. Mercier F, Kitasako JT, Hatton GI. Anatomy of the brain neurogenic zones revisited: fractones and the fibroblast/macrophage network. J Comp Neurol 2002 2002-09-16;451(2):170-188.
14. Kim JT, Park MS, Choi KH, et al. The CBV-ASPECT Score as a predictor of fatal stroke in a hyperacute state. Eur Neurol.2010;63(6):357-63.
1.吴兆苏,姚崇华,赵冬.我国人群脑卒中发病率、死亡率的流行病学研究.中华流行病学杂志.2003；24：236-239。
2.刘承基.脑血管外科学.南京：江苏科学技术出版社;
3. Gialanella B, Ferlucci C. Functional outcome after stroke in patients with aphasia and neglect:assessment by the motor and cognitive functional independence measure instrument. Cerebrovasc Dis 2010 2010-01-20;30(5):440-447.
4. Miiller HD, Hanumanthiah KM, Diederich K, et al. Brain-derived neurotrophic factor but not forced arm use improves long-term outcome after photothrombotic stroke and transiently upregulates binding densities of excitatory glutamate receptors in the rat brain. Stroke.2008, 39(3):1012-1021. Epub 2008 Jan 31.
5.杨小锋,虞军,刘伟国,等。脑源性神经营养因子和神经干细胞联合移植治疗大鼠重度颅脑损伤的研究.浙江医学,2005,27：1-3.
6. Kermani P, Hempstead B. Brain-derived neurotrophic factor:a newly described mediator of angiogenesis. Trends Cardiovasc Med.2007,17(4):140-143.
7.黄东煜,张志坚,陈柏龄,等。慢病毒载体介导Bdnf基因修饰的骨髓间质干细胞移植治疗脑梗死。生物工程学报,2008；24(7)：1174-1179.
8. Li B, Piao CS, Liu XY, Guo WP, Xue YQ, Duan WM, et al. Brain self-protection:the role of endogenous neural progenitor cells in adult brain after cerebral cortical ischemia. Brain Res 2010 2010-04-23;1327:91-102.
9. Zhang Y, Pardridge WM. Neuroprotection in transient focal brain ischemia after delayed intravenous administration of brain-derived neurotrophic factor conjugated to a blood-brain barrier drug targeting system. Stroke 20012001-06-01;32(6):1378-1384.
10. Yanamoto H, Nagata I, Sakata M, Zhang Z, Tohnai N, Sakai H, et al. Infarct tolerance induced by intra-cerebral infusion of recombinant brain-derived neurotrophic factor. Brain Res 2000 2000-03-24;859(2):240-248.
11. Schabitz WR, Schwab S, Spranger M, Hacke W. Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats. J Cereb Blood Flow Metab 1997 1997-05-01;17(5):500-506.
12. Han Q, Sun W, Lin H, Zhao W, Gao Y, Zhao Y, et al. Linear ordered collagen scaffolds loaded with collagen-binding brain-derived neurotrophic factor improve the recovery of spinal cord injury in rats. Tissue Eng Part A 2009 2009-10-01;15(10):2927-2935.
13.8.Teng H, Zhang ZG, Wang L, et al. Coupling of angiogenesis and neurogenesis in cultured endothelial cells and neural progenitor cells after stroke. J Cereb Blood Flow Metab.2008, 28(4):764-771.
14. Yang YJ, Wang XL, Yu XH, et al. Hyperbaric oxygen induces endogenous neural stem cells to proliferate and differentiate in hypoxic-ischemic brain damage in neonatal rats. Undersea Hyperb Med.2008,35(2):113-129.
15.林百喜,谢明,武衡。大脑中动脉阻塞大鼠脑内脑源性神经营养因子蛋白表达变化。中国现代医学杂志,2008,18：702-705.
16. Pencea V, Bingaman KD, Wiegand SJ, et al. Infusion of brain—derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypOthalamusfJ. J Neurosci,2001,21(17):6706-6717.
17. Kim MW, Bang MS, Han TR,et al. Exercise increased BDNF and trkB in the conrralateral hemisphere of the ischemic rat brain. Brain Res.2005,1052(1):16-21.
18.汪志凌,毛萌,周晖,等。脑源性神经营养因子对缺氧胚脑神经元保护作用的信号传递途径初探。四川大学学报(医学版),2007,38：934-937.
19. Sun C, Hu Y, Chu Z, et al. The effect of brain-derived neurotrophic factor on angiogenesis. J Huazhong Univ Sci Technolog Med Sci.2009,29(2):139-143.
20.孙春艳,胡豫,,吴涛,等。脑源性神经营养因子对血管新生的作用.中华病理学杂志,2006,35：238-239.
21. Leventhal C, Rafii S, Rafii D, Shahar A, Goldman SA. Endothelial trophic support of neuronal production and recruitment from the adult mammalian subependyma. Mol Cell Neurosci 1999; 13:450-464.
22. Fu YK, Chang CJ, Chen KY, et al. Imaging of regional metabolic activity by (18)F-FDG/PET in rats with transient cerebral ischemia. Appl Radiat Isot.2009 Oct;67(10):1743-7.
23.郭拮,王瑞民,张锦明,等。阿尔茨海默病大鼠模型PET在体成像研究。首都医科大学学报,2008,29：12-14.
24.8. Jiang Y, Jahagirdar BN, Reinhardt RL. et al. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature,2002,418(6 893):41
1. Kim JT, Park MS, Choi KH, et al. The CBV-ASPECT Score as a predictor of fatal stroke in a hyperacute state. Eur Neurol. 2010; 63 (6):357-63.
2.李培勇,郭万华等.SPM和ROI方法研究脑内?99Tc?m—ECD的稳定性.中华核医学杂志2001；21(4)：235-236
3. Chen R, Liang F, Ishigami K, et al. Clinical risk factors in regional brain ischemia using single photon emission computed tomography. J Am Geriatr Soc.2010;58(7):1411-2.
4. Kiewert C, Mdzinarishvili A, Hartmann J, et al. Metabolic and transmitter changes in core and penumbra after middle cerebral artery occlusion in mice. Brain Res.2010;1312:101-107.
5. Shimosegawa E, Hatazawa J, Inugami A, et al. Cerebral infarction within six hours of onset:prediction of completed infarction with technetium-99m-HMPAO SPECT. J Nucl Med.1994;35(7):1097-1103.
6. Uyttenboogaart M, De Keyser J, Luijckx GJ. Thrombolysis for acute ischemic stroke. Curr Top Med Chem 2009;9(14):1285-1290.
7. Abels B, Klotz E, Tomandl BF, et al. Perfusion CT in acute ischemic stroke:a qualitative and quantitative comparison of deconvolution and maximum slope approach. AJNR Am J Neuroradiol. 2010;31(9):1690-1698.
8. Nuutinen J, Liu Y, Laakso MP, et al. Perfusion differences on SPECT and PWI in patients with acute ischemic stroke. Neuroradiology. 2009;51(10):687-695.
9. Langheinrich AC, Yeniguen M, Ostendorf A, et al. Evaluation of the middle cerebral artery occlusion techniques in the rat by in-vitro 3-dimensional micro- and nano computed tomography. BMC Neurol. 2010;10:36.
10. Liu Z, Barrett HH, Stevenson GD, et al. Evaluating the protective role of ischaemic preconditioning in rat hearts using a stationary small-animal SPECT imager and 99mTc-glucarate. Nucl Med Commun.2008;29(2):120-128.
[1]Spiegelstein 0, Eudy JD, Finnell RH. Identification of two putative novel folate receptor genes in humans and mouse[J], Gene,2000,258 (1-2):117-25.
[2]Antony AC. Folate receptors[J]. Ann Rev Nutr,1996;16:501-21.
[3]Weitman SD, Frazier KM, Kamen BA. The folate receptor in central nervous system malignancies of childhood. Neurooncol,1994,21:107-112.
[4]Todd A. Patrick, David M. Kranz, Terry A. van Dyke et al. Folate receptors as potential therapeutic targets in choroid plexus tumors of SV40 transgenic mice[J]. Journal of Neuro-Oncology,1997,32:111-123.
[5]Oyesiku NM, Evans CO, et al. Differential expression of folate receptor in pituitary adenomas[J]. Cancer Res,2003,63:4218-4224.
[6]Ladino CA, Chari RV, Bourret LA, et al. Folate—maytansinoids: target—selective drugs of low molecular weight[J]. International journal of cancer,1997,73:859-864.
[7]Aronov o, Horowitz AT, Gabizon A, et al. Folate—targeted PEG as a potential carrier for Carboplatin analogs[J]. Synthesis and in vitro studies, Bioconjugate chemistry,2003,14:563-574.
[8]Reddy JA, Dorton R, Westrick E, et al. Preclinical evaluation of EC145, a folate-vinca alkaloid conjugate[J]. Cancer Res 2007,67:4434-4442.
[9]ZHAOX B, LEE R J, et al. Tumor-slectivetargeted delivery of genes and antisense oligodeoxyribonucleotides via the folate receptor[J]. Adv Drug Ddiv Rev,2004,56(8):1193-1204.
[10]Lu Y, Low PS, et al. Folate targeting of haptens to cancer cell surfaces mediated immunotherapy of syngenic murine tumors[J]. Cancer Immunol Immunother,2002,51:153-162.
[11]Siegel BA, Dehdashti F, Mutch DG, et al. Evaluation of 111In-DTPA-folate as a receptor-targeted diagnostic agent for ovarian cancer:initial clinical results[J].J Nucl Med 2003,44:700-707.
[13]Kalli KR, Oberg AL, Keeney GL, et al. Folate receptor alpha as a tumor target in epithelial ovarian cancer. Gynecol Oncol 2008,108:619-626.
[14]Yamamoto T, Tsuboi K. Particle radiotherapy for malignant gliomas. Brain Nerve 2009,;61 (7):855-66.
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