活化小胶质细胞在急性脑梗死中的作用及其机制的研究
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摘要
研究背景与现状
脑血管疾病(cerebrovascular disease, CVD)作为神经系统较为常见的疾病,是主要的公共健康问题之一。脑梗死又称缺血性卒中,是指各种原因所致脑部血液供应障碍,导致脑组织缺血、缺氧性死亡,并出现相应的神经功能缺损的疾病。脑梗死是脑血管病的常见类型,约占全部脑血管病的80%,是继心脏病后又一种高致死性的疾病。既使有些病人幸存下来也遗留了严重的后遗症,给家庭和社会带来沉重的负担,而随着老龄化社会的到来,缺血性脑病的发病率也在逐年增加,因此,寻找一种安全而有效的治疗方法成为当今医学界研究的热点内容。关于脑梗死的病理变化,目前多数学者认为脑部的急性缺血缺氧所导致的小胶质细胞的活化,炎症因子、兴奋性氨基酸的释放,氧自由基的产生等这些因素是造成脑损伤的重要原因,但具体的作用机制仍不清楚。小胶质细胞(Microglia, MG)是脑内主要的免疫细胞,具有营养、保护和修复神经元的作用。静止的MG发挥了免疫监视作用,而活化的MG在中枢神经系统疾病中的作用仍存在较大的争议。以前大量的体外研究显示MG在中枢神经系统疾病中发挥着神经损害的作用,而近些年来的体内研究却显示活化的MG不仅分泌神经营养因子,还抑制了神经元的凋亡,因而具有神经保护作用。因此,明确小胶质细胞在脑梗死中的作用将有助于解释该病的病理过程并且有助于寻找新的治疗方法,所以有必要对MG功能进行深入研究,以期明确活化MG在神经系统疾病中的确切作用及机制。
细胞外调节蛋白激酶(extracellular signal-regulated kinases, ERK)分为ERK1和ERK2,统称为ERK1/2。ERK是一丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)家族中的一个重要成员,广泛地参与了从细胞的代谢、活力及炎症反应到细胞死亡与生存的调节。脑梗死后的生长因子或细胞因子的刺激可以经细胞表面的三级MAPK的模式将其磷酸化而激活,活化后的ERKl/2大部分转移至细胞核内,促进某些基因的转录与表达,与细胞的增殖与分化密切相关。许多研究揭示磷酸化的ERK1/2主要表达于脑梗死后梗死皮层周围的神经元内,也有文献报道脑缺血的刺激可引起MG内的ERK1/2磷酸化,活化后的ERK1/2促进了MG合成和分泌BDNF。
脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)是神经营养因子家族的成员之一,除分布于中枢神经和感觉神经以外,还分布于脊髓的运动神经元等。由于人们认识到BDNF在神经疾病中的重要作用,所以对其的功能进行了广泛的研究,许多研究结果提示BDNF不仅对神经元的生存、增殖、生长与分化以及维持正常的神经元功能起着重要的作用,而且还增加了神经元对伤害性刺激的耐受性,促进神经元再生等作用,但目前更多学者公认的BDNF神经保护机制是关于BDNF在拮抗兴奋性氨基酸毒性,稳定细胞内钙离子浓度中的作用。研究表明BDNF可抑制Ca2+内流和细胞内钙离子的释放,BDNF可增加在海马内的含钙结合蛋白的神经元数量,而含钙结合蛋白的神经元较不含钙结合蛋白的神经元具有更好的抵抗谷氨酸毒性和抑制细胞内Ca2+升高的能力,因此BDNF可通过增加细胞内钙结合蛋白来稳定细胞内钙离子的浓度。
Calbindin-D28k是钙结合蛋白的重要成员,其在脑血管疾病中的作用日益受到人们的重视,许多研究发现Calbindin-D28k存在于神经元中,却没有参与神经系统的信号传递,提示该蛋白通过对细胞外钙离子浓度的调节参与了神经元的某些生理功能,可能在突触的联系中发挥了一定的作用,使神经元免于遭受由电压差所引起的细胞内钙超载；Calbindin-D28k可与钙离子高亲和力地结合从而缓冲了钙离子在细胞内快速地增加,并参与了钙离子的运输,阻止钙离子过度地蓄积,从而保持了钙离子浓度的相对稳定。这些结果均提示Calbindin-D28k在脑损伤后通过抑制钙离子的浓度过高而发挥神经保护作用。另外,进一步的研究发现Calbindin-D28k能激活Ca2+/Mg2+-ATP酶活性,阻止脑内Ca2十过度蓄积。Calbindin-D28k缓冲神经元内过高的钙离子浓度和运输细胞内异常升高的Ca2+是维持钙离子稳态不可缺少的重要因素。还有很多文献报道钙结合蛋白通过它的缓冲钙的作用,能够显著地减轻缺血缺氧引起的神经细胞的损伤。另外,Calbindin-D28k另一个神经保护机制可能与与抑制钙蛋白酶活性有关,因为在急性脑梗死后,大量的钙离子开始进入神经细胞内,从而激活了钙蛋白酶。活化的钙蛋白酶可将神经元内的膜收缩蛋白(a-spectrin,神经元的基本结构单位)水解为分子量为145和150KDa的蛋白质片段,从而造成神经元的坏死。
既然MG在缺血性脑病中发挥了重要作用,而其在脑梗死特别是急性脑梗死中的作用及其作用机制仍不清楚,因此有必要在体内进一步研究MG的作用及作用的分子机制,以期为脑梗死的治疗提供新的治疗途径与靶点。
目的：为体内研究活化小胶质细胞在急性脑梗死中的作用提供纯化的种子细胞。
方法：取胎龄14天的昆明小鼠大脑,采用改良的胰蛋白酶预消化加轻拍打法提取和纯化小胶质细胞,然后采用免疫细胞化学方法检测细胞质表面的抗原标记,采用流式细胞技术检测细胞的纯度。将经过纯化的小胶质细胞进行传代培养。
结果：原代培养的结果显示经改良后的McCarthy法即胰蛋白酶预消化加轻拍打法所纯化的细胞数量较多,且细胞免疫化学法检测显示多为CD68和CDllb阳性细胞,流式细胞技术检测显示改良后的胰蛋白酶预消化加轻拍打法所收集到的CDllb阳性细胞所占细胞总数的百分比超过了92%,而传统的McCarthy方法收集的CDl1b阳性细胞纯度只有80%,并且两种纯化方法所收集的CD11b阳性细胞的纯度有显著的统计学差异(p<0.05),因此,经改良的McCarthy方法分离纯化的细胞达到了实验要求,可以作为种子细胞进行以下实验。
结论：胰蛋白酶预消化加轻拍打法可以分离纯化到纯度较高、数量较多的小胶质细胞。
目的：体内研究活化小胶质细胞在大体水平对脑梗死急性期小鼠的神经功能和梗死体积的影响。
方法：采用羧基荧光素二乙酸盐琥珀酰亚胺酯(CFDA-SE)液对小胶质细胞进行标记,并于标记后12、24和72h及7d在荧光显微镜下观察荧光信号,随后用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法检测CFDA-SE对小胶质细胞生长及增殖的影响。采用线栓栓塞大脑中动脉(MCAO)方法制做脑梗死动物模型；在观察荧光信号达到实验要求的72h后,将CFDA-SE标记的小胶质细胞经锁骨下静脉注入到MCAO小鼠体内,并分别于移植后的12、24和72h取小鼠大脑做冰冻切片以观察小胶质细胞在脑内的分布与数量。完成上述的细胞标记与示踪实验后,将66只清结级昆明小鼠随机分为四组：A组(MCAO+小胶质细胞移植,小胶质细胞移植组),B组(MCAO+MG培养基,安慰剂组),C组(MCAO组,空白对照组),D组(假手术+小胶质细胞移植,假手术组),其中A组MCAO小鼠在术后12h接受50ì1小胶质细胞悬液(1×105/μ1)；B组MCAO小鼠在术后12h接受与A组相同体积的培养基；C组小鼠在经MCAO术未接受任何处理；D组小鼠在经假手术12h后接受了与A组相同体积的小胶质细胞悬液。然后分别于小胶质细胞移植后的12、24和72h采用Zea-longa和圆筒试验对各组小鼠进行神经功能评分,随后处死小鼠后取脑,采用2%红四氮唑(TTC)法测量脑梗死的体积。
结果：细胞标记的结果显示：于CFDA-SE标记后的12h可在荧光显微镜下观察到带有很强绿色荧光信号的小胶质细胞,并且该荧光信号一直持续到第7天,说明该标记液达到了本实验的要求,另外,MTT法检测显示CFDA-SE并没有影响小胶质细胞的生长与增殖。脑组织的冰冻切片显示在移植后的12h,带有荧光信号的小胶质细胞开始在MCAO小鼠脑内出现,在移植后的72h明显增多,并且主要集中在脑梗死周围。神经功能评定显示与安慰剂B组和空白对照C组相比,MG移植A组小鼠Zea-longa评分降低,患侧前肢首次接触瓶壁的次数也明显增加,并且在统计学上有显著的差异(p<0.05)。TTC染色显示A组小鼠的梗死体积显著小于B和C组小鼠(p<0.05)。
结论：CFDA-SE标记液能够对小胶质细胞进行绿色荧光标记,并且标记的荧光信号能够维持到标记后的第7天而没有影响小胶质细胞的生长与增殖；体外移植的带有绿色荧光信号的小胶质细胞能够通过血脑屏障到达MCAO小鼠脑内,并且在移植后72h在脑梗死周围明显增多；体外移植的活化小胶质细胞改善了MCAO小鼠前肢的神经功能,减小了梗死体积。
目的：在组织及分子水平研究小胶质细胞是否影响了脑梗死急性期神经元的死亡和相关细胞因子的释放。
方法：25-35g清洁级雄性昆明小鼠264只。动物的分组及处理同第二章的实验内容。分别于小胶质细胞移植后的12h、24h和72h采用过量麻醉方法处死小鼠并取脑,然后采用HE染色法检测坏死的神经细胞,采用TUNEL染色法检测凋亡的神经细胞,采用免疫组织化学方法检测脑源性神经营养因子(BDNF)、胶质源性神经营养因子(GDNF)、肿瘤坏死因子(TNF-α)和白细胞介素-1β(IL-1β)等细胞因子及微管相关蛋白(MAP2)的表达,采用免疫荧光双染法检测了磷酸化的细胞外信号调节激酶(p-ERK1/2)在神经细胞、小胶质细胞和星型胶质细胞内的表达,另外,采用western blot方法检测了神经细胞的裂解的膜收缩蛋白(cleaved α-spectrin,神经元的骨架结构)的表达。
结果：在小胶质细胞移植后的24和72h与B和C组小鼠相比,A组小鼠的HE阳性细胞数量明显减少,而TUNEL阳性细胞、GDNF、TNF-α和IL-1β阳性细胞却无显著的增加或减少,BDNF和MAP2的阳性细胞数量显著地增加,另外,cleaved α-spectrin(分子量为145KDa)的表达水平也明显升高。免疫荧光双染显示p-ERK1/2除少量表达于小胶质细胞外,还大量地表达于神经细胞核。
结论：活化小胶质细胞抑制了神经细胞的坏死并有助于神经细胞的存活,促进了BDNF及p-ERK1/2的表达,但并没有影响神经细胞的调亡和TNF-a、IL-1β和GDNF的表达。
目的：研究活化小胶质细胞在脑梗死急性期对P-ERK1/2表达的影响及其影响的意义,为活化小胶质细胞的神经保护机制提供了理论依据。
方法：198只清洁级昆明小鼠被随机分为四组：A组(MCAO+小胶质细胞移植,小胶质细胞移植组),B组(MCAO+小胶质细胞移植+U0126,U0126抑制剂组),C组(MCAO,对照组),D组(假手术组),其中A和B组MCAO小鼠在术后12小时接受50μl小胶质细胞悬液(1×105细胞/μ1)的移植；B组小鼠在术后接受一定剂量的U0126溶液；C组小鼠在手术后未接受任何处理。在建立脑梗死或者假手术模型后,立即将U0126溶液按照30mg/kg的剂量经腹腔注入B组小鼠,以后每6小时注射1次直到实验完成,以维持一定的血药浓度。分别于小胶质细胞移植后的12、24和72h取脑,采用免疫组化方法检测BDNF阳性细胞数量,采用RT-PCR方法在基因水平检测BDNF及钙结合蛋白酶D28K(Calbindin-D28K)在各组的表达,采用western-blot方法检测了P-ERK1/2、ERK1/2、BDNF、Calbindin-D28k和cleaved-a-spectrin的表达。
结果：与B和C组小鼠相比,A组小鼠脑内的p-ERK1/2的蛋白表达水平和BDNF阳性细胞数量在移植后24和72h显著增加(P<0.05)。Rt-PCR和western-blot检测结果显示A组小鼠脑内BDNF的表达水平在移植后24和72h显著高于B和C组小鼠(P<0.05),但A组Calbindi-D28k表达的时间较晚,在移植后72h才与B和C组有显著差异(P<0.05)。另外,在移植后的72h,A组小鼠脑内a-spectrin(分子量为145KDa)裂解片段的蛋白表达水平显著低于B和C组(P<0.05),而B和C组小鼠脑内a-spectrin(分子量为145KDa)裂解片段的蛋白表达水平却无显著差异(P>0.05)。
结论：活化小胶质细胞在脑梗死急性期通过p-ERK1/2信号通路促进了神经细胞内的BDNF的表达,进而BDNF促进了Calbindin-D28k蛋白的表达,最终抑制了神经细胞的坏死。
Background
Cerebrovascular disease (CVD), served as a common disease of central neurvous system (CNS), is a major issue of public health. Cerebral infarct, also named ischemic stroke, is the disease that the death of hypoxic and ischemic brain tissue resulting from the disorder of blood supply leads to the symptom of neurological deficits. Cerebral infarct, which is the most common type of CVD and accounts for80%of the incidence, is another high mortility disease followed ischemic heart disease. Even so some patiens survives from the disease, they remains severel disabled, thus imposing a heavy burden to fomily and community. In addition, the incidence is predicted to grow with the increasing age of the population. Therefore, finding a safe and effective therapeutic method has been major purpose of medicine. At present, most studies indicated that brain injury attributed to those factors such as microglial activity under cerebral hypoxia and ischemical condition, release of proinflammar factors and excitory amino acids and generation of oxygen free radicals. However, the exact mechanisms remain unclear. MG are major immunocytes and play neurotrophic, neuroprotective and repaireing neuron cells role. Rest MG provide immuno-minitor role but the effect of activated MG in central nervous disease (CND) is still debated. Previous in vitro studies displayed that activated MG provide a neuroprotective role in CND through releasing neurotrophic factors or inhibit neurons apoptosis. Therefore, identifying the effect of activated MG after cerebral infarct would contribute to explain the pathological process of cerebral infarct and to find new therapeutic method. Therefore, it is necessary that further studying role of MG in CND in order to reveal the exact effect and mechanism of activated MG
Extracellular signal-regulated kinase1/2(ERK1/2), one of the best-characterized members of the mitogen-activated protein kinase (MAPK) family, mediates a range of activity from metabolism, motility, and inflammation to cell death and survival. It is phosphorylated and activated through a three-tiered MEK mode via cell surface receptors stimulated by growth factors or cytokines. Most phosphorylated ERK1/2translocates to cell nucleus and induces genic transduction and expression and is closely associated with cell proliferation and differentiation. It is well documented that phosphor-ERK1/2mainly expresses in neurons of cortex in peri-infarct sites after acute cerebra ischemia. It is also reported that ERK1/2located in MG is phosphorylated in response to acute cerebral infarct and phosphor-ERK1/2promotes MG to synthesize and secrete brain-derived neurotrophic factor (BDNF).
BDFN is a member of neurotrophic factor family and generally locals in CNS, sensory neurons and the neurons of spinal cord motor. Recently, numerous studies from world have confirmed that BDNF not only play a critical role in neuron surviva, differentiation and sustaining normal physiological function but protecting neurons against injury. Meanwhile, it also promotes neuron regeneration. However, more views supported that the neuroprotective roles of BDNF are due to inhibiting cytotoxity of excitory amino acids and stabling Ca2+concentration of cytoplasm. Some studies have showed that BDNF prevented Ca2+from entering into cell and secreting from cytoplasm. The BDNF treatment increases the number of hippocampus neuron with calcium binding protein (CBP). The neurons with CBP show more significant ability in resisting glumatic acid cytotoxity and reducing Ca2+concentration in cytoplasm than neurons not with CBP. Therefore, BDNF could stable Ca2+concentration in cytoplasm through CBP role.
Calbindin-D28k is a major member of CBP family. Recently, many experts have done numerous in vivo and in vitro studies to explore the effect of calbindin-D28k. although calbindin-D28k locates in neurons, it is not linked with neurotransmitter at all. It indicated that calbindin-D28k role in mediating Ca2+concentration in brain is associated with the some phosiological repense of neurons with CBP. Calbindin-D28k plays a role in synaps linking and maybe prevents neurons subjecting to injury due to excessive Ca2+concentration-depended on voltage difference. Calbindin-D28k stabilizes Ca2+concentration in cytoplasm through stably binding Ca2+and buffering and transporting Ca2+to arrest over Ca2+accumulation. Calbindin-D28k can activate Ca2+/Mg2+/-ATP enzyme activation and arrest Ca2+excessive accumulation in cytoplasm. It is a critical factor in sustaining Ca2+balance that calbindin-D28k bufferes and transports excessive Ca2+in neuronal plasm. Many studies have revealed that calbindin-D28k can remarkly reduce neurons injury induced by hypoxia and ischemia. In addition to this, the neuroprotective role of Calbindin-D28k maybe be associated with inhibiting calpain activation. Excessive elevation of cytosolic Ca2+levels after acute cerebral infarct activates calpain. Activated calpain can cleave a-spectrin into two protein fragments as molecular weight of145and150KDa and neurons necrosis is increased.
Since MG play a critical role in ischemic cerebral injury and the effect and molecular mechanisms of atctivated MG in response to cerebral infarct especially actue cerebral infarct remain unclear, it is necessary to further study the role of activated microglia under in vivo condition after acute cerebral ischemia. The present study maybe provide new method and potential target for treating acute cerebral infarct.
Objective:To provide transplanting microglia for detecting the effect of activated microglia after acute cerebral ischemia.
Methods:Mixed glial cells were obtained from cortex of SPF Kunming mouse embryo for14days. Traditional McCarthy and modulated McCarthy method i.e trypsin predigesting combinated with slight tapping were employed to extract and purify MG Then immunocytochemistry was used to detect the antigenic markers of cell and flow cytometry was employed to determined cultured cell purity. Finally, purity cell was cultured or subcultured.
Results:Primary cultured results showed that more cells were obtained by modified McCarthy method than traditional McCarthy method. Immunocyto chemistry determining showed that the surface antigen of most puriftied cells was CD68and CDllb. the results of flow cytometry technique showed the number of CD11b positive cells was over92%of total cells number by modilated method but80%by McCarthy and there was significant difference between modilated and traditional McCarthy methods in the number of CDllb positive cells (p<0.05). Therefore, these cells by isolating and puritfy were microglia and can be employed to the studies of microglial function.
Conclusions:Modified McCarthy method improves microglial yield and purity.
Objective:To determine effect of activated microglia on neurological function and infarct volume of mouse after acute cerebral ischemia in vitro.
Methods:First, MG were marked by CFDA-SE with green fluorescence and the signal intension of fluorescence was observed under fluorescence microscope at12,24and72h as well as7day after marking. In addition, MTT was employed to detect the effect of CFDA-SE on the growth and proliferation of MG. Secondly, Once the signal intension reached the experiment criterion, MG marked by CFDA-SE were injected into permenant middle cerebral artery occlusion (pMCAO) mice body through subclavian vein, and the brains were disassociated at12,24and72h. The disassociated brains were sliced by frost slice techniques and the number of MG with fluorescence was observed under fluorescence. Finally,66mice were divided randomly into4groups:A group (MCAO+MG transplanting), B group (MCAO+MG medium), C group (MCAO group) and D group (sham operation+MG transplanting). Triphenyltetrazolium chloride (TTC) was employed to detect infarct volume and Zea-longa and cylinder test were used to evaluate neurological deficits at12,24and72h after transplanting.
Results:The results derived from cell marking showed that the flueroscence intension of marked cells was significant and unchanged at at12,24and72h after treatment. However, the signal intension of attached cells was partially attenuated at7day after treatment. MTT test revealed that CFDA-SE did not affect the growth and proliferation of microglia. The results derived from cell tracing showed that few cells marked with flueroscence was observed at12h after transplanting and the number of marked cells was increased at24and further increased at72h after treatment. In addition, the marked cells mainly scattered in peri-infarct site at72h after treatment. Compared with B and C groups, mice in A group had smaller infarct volume, lower Zea-longa score and more frequencies touching bottle wall with hemi-paretic forepaws at24and72h after transplanting (P<0.05).
Conclusions:CFDA-SE marking liquid is a good liquid marking MG and dose not affect MG growing and proliferation. CFDA-SE sustains intensive flueroscene signal for7days and it has met the criterion of present experiment. Primary cultured MG can enter the peri-infarct site of MCAO mice through blood brain barrier and the number of MG in brain is increased at72h after transplanting. Activated microglia play a neuroprotective role generally after acute cerebral ischemia.
Objective:To detect whether activated microglia influence neuronal death and releasing of cytokines after acute cerebral ischemia in tissue and molecular levels.
Methods:Two hundred sixty four adult male SPF kunming mouse weighing25-35g were used in present section. The animal grouping and treating were similar with Part Ⅱ. Nectrotic and apoptosic neuron cells, survival neurons and proinflammar and neurotrophic factors, such as TNF-a, INL-1β,GDNF and BDNF, in brain of mice in4groups were determined by HE, TUNEL, immunohistochemistry at12,24and72h after transplanting. Duable flueronscence staining was used to detect phosphorylated ERK1/2expression on MG, astrocytes and neurons. Western blot was used to test the expression levels of cleaved a-spectrin.
Results:Compared with B and C groups, mice in A group showed less HE positive cells but similar number in TUNEL positive cells in peri-infarct at24and72h after transplanting (P<0.05). There were no difference among the three groups in TNF-a, IL-1β and GDNF positive cells number at24and72h after treatment (P>0.05). However, the number of BDNF positive cells was remarkly increased compared with B and C groups (P<0.05). Duable flueroscene staining showed that pERKl/2mainly located in neuronal nuclear in the brain of MCAO or sham-operation mice. Compared with B, C and D group, the number of pERK1/2positive cells was remarkly increased at24and72h after transplantion. In adddition, there was no difference between B and C groups in the number of pERKl/2positive cells (P>0.05).
Conclusions:Activated microglia inhibit neuronal cells necrosis and promote BDNF and p-ERK1/2expressions after acute cerebral ischemia.
Objective:To determine the effect of activated microglia on the expression of p-ERK1/2and significance of the effect after acute cerebral ischemia, and to provide theory evidence.
Methods:Two hundred and sixteen mice were divided randomly into4groups: A group (MCAO+MG transplanting), B group (MCAO+MG transplanting+U0126, inhibitor group), C group (MCAO group) and D group (sham operation). Western blot was used to test the expression levels of pERKl/2, BDNF, calbindin-D28k and cleaved a-spectrin at12,24and72h after acute cerebral infarct. Immunohistochemistry and RT-PCR were used to detect the number of BDNF positive cells and the expression level of BNDF and calbindin-D28k mRNA respectively at the three time points.
Results:Compared with B and C groups, expression levels of pERK1/2protein and the number of BDNF positive cells of mice in A group were increased at24and72h after transplanting (P<0.05). The expression levels of BDNF mRNA and protein were increased at24and72h after transplanting and the increase in expression level of calbindin-D28k protein was at72h (P<0.05). In addition, the expression level of cleaved a-spetrin protein in A group was remarkably increased compared with B and C group at72h after treatment (P<0.05). However, there was no difference between B and C groups in the expression of pERKl/2, BDNF, calbindin-D28k and cleaved a-spectrin protein and mRNA expressions of BDNF and calbindin-D28k (P>0.05).
Conclusions:Activated microglia up-regulate expression of BDNF through ERK1/2signal pathway. BDNF inhibit neuronal necrosis through promoting expression of Calbindin-D28K after acute cerebral ischemia.
脑血管疾病(cerebrovascular disease, CVD)作为神经系统较为常见的疾病,是主要的公共健康问题之一。脑梗死又称缺血性卒中,是指各种原因所致脑部血液供应障碍,导致脑组织缺血、缺氧性死亡,并出现相应的神经功能缺损的疾病。脑梗死是脑血管病的常见类型,约占全部脑血管病的80%,是继心脏病后又一种高致死性的疾病。既使有些病人幸存下来也遗留了严重的后遗症,给家庭和社会带来沉重的负担,而随着老龄化社会的到来,缺血性脑病的发病率也在逐年增加,因此,寻找一种安全而有效的治疗方法成为当今医学界研究的热点内容。关于脑梗死的病理变化,目前多数学者认为脑部的急性缺血缺氧所导致的小胶质细胞的活化,炎症因子、兴奋性氨基酸的释放,氧自由基的产生等这些因素是造成脑损伤的重要原因,但具体的作用机制仍不清楚。小胶质细胞(Microglia, MG)是脑内主要的免疫细胞,具有营养、保护和修复神经元的作用。静止的MG发挥了免疫监视作用,而活化的MG在中枢神经系统疾病中的作用仍存在较大的争议。以前大量的体外研究显示MG在中枢神经系统疾病中发挥着神经损害的作用,而近些年来的体内研究却显示活化的MG不仅分泌神经营养因子,还抑制了神经元的凋亡,因而具有神经保护作用。因此,明确小胶质细胞在脑梗死中的作用将有助于解释该病的病理过程并且有助于寻找新的治疗方法,所以有必要对MG功能进行深入研究,以期明确活化MG在神经系统疾病中的确切作用及机制。
细胞外调节蛋白激酶(extracellular signal-regulated kinases, ERK)分为ERK1和ERK2,统称为ERK1/2。ERK是一丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)家族中的一个重要成员,广泛地参与了从细胞的代谢、活力及炎症反应到细胞死亡与生存的调节。脑梗死后的生长因子或细胞因子的刺激可以经细胞表面的三级MAPK的模式将其磷酸化而激活,活化后的ERKl/2大部分转移至细胞核内,促进某些基因的转录与表达,与细胞的增殖与分化密切相关。许多研究揭示磷酸化的ERK1/2主要表达于脑梗死后梗死皮层周围的神经元内,也有文献报道脑缺血的刺激可引起MG内的ERK1/2磷酸化,活化后的ERK1/2促进了MG合成和分泌BDNF。
脑源性神经营养因子(brain-derived neurotrophic factor, BDNF)是神经营养因子家族的成员之一,除分布于中枢神经和感觉神经以外,还分布于脊髓的运动神经元等。由于人们认识到BDNF在神经疾病中的重要作用,所以对其的功能进行了广泛的研究,许多研究结果提示BDNF不仅对神经元的生存、增殖、生长与分化以及维持正常的神经元功能起着重要的作用,而且还增加了神经元对伤害性刺激的耐受性,促进神经元再生等作用,但目前更多学者公认的BDNF神经保护机制是关于BDNF在拮抗兴奋性氨基酸毒性,稳定细胞内钙离子浓度中的作用。研究表明BDNF可抑制Ca2+内流和细胞内钙离子的释放,BDNF可增加在海马内的含钙结合蛋白的神经元数量,而含钙结合蛋白的神经元较不含钙结合蛋白的神经元具有更好的抵抗谷氨酸毒性和抑制细胞内Ca2+升高的能力,因此BDNF可通过增加细胞内钙结合蛋白来稳定细胞内钙离子的浓度。
Calbindin-D28k是钙结合蛋白的重要成员,其在脑血管疾病中的作用日益受到人们的重视,许多研究发现Calbindin-D28k存在于神经元中,却没有参与神经系统的信号传递,提示该蛋白通过对细胞外钙离子浓度的调节参与了神经元的某些生理功能,可能在突触的联系中发挥了一定的作用,使神经元免于遭受由电压差所引起的细胞内钙超载；Calbindin-D28k可与钙离子高亲和力地结合从而缓冲了钙离子在细胞内快速地增加,并参与了钙离子的运输,阻止钙离子过度地蓄积,从而保持了钙离子浓度的相对稳定。这些结果均提示Calbindin-D28k在脑损伤后通过抑制钙离子的浓度过高而发挥神经保护作用。另外,进一步的研究发现Calbindin-D28k能激活Ca2+/Mg2+-ATP酶活性,阻止脑内Ca2十过度蓄积。Calbindin-D28k缓冲神经元内过高的钙离子浓度和运输细胞内异常升高的Ca2+是维持钙离子稳态不可缺少的重要因素。还有很多文献报道钙结合蛋白通过它的缓冲钙的作用,能够显著地减轻缺血缺氧引起的神经细胞的损伤。另外,Calbindin-D28k另一个神经保护机制可能与与抑制钙蛋白酶活性有关,因为在急性脑梗死后,大量的钙离子开始进入神经细胞内,从而激活了钙蛋白酶。活化的钙蛋白酶可将神经元内的膜收缩蛋白(a-spectrin,神经元的基本结构单位)水解为分子量为145和150KDa的蛋白质片段,从而造成神经元的坏死。
既然MG在缺血性脑病中发挥了重要作用,而其在脑梗死特别是急性脑梗死中的作用及其作用机制仍不清楚,因此有必要在体内进一步研究MG的作用及作用的分子机制,以期为脑梗死的治疗提供新的治疗途径与靶点。
目的：为体内研究活化小胶质细胞在急性脑梗死中的作用提供纯化的种子细胞。
方法：取胎龄14天的昆明小鼠大脑,采用改良的胰蛋白酶预消化加轻拍打法提取和纯化小胶质细胞,然后采用免疫细胞化学方法检测细胞质表面的抗原标记,采用流式细胞技术检测细胞的纯度。将经过纯化的小胶质细胞进行传代培养。
结果：原代培养的结果显示经改良后的McCarthy法即胰蛋白酶预消化加轻拍打法所纯化的细胞数量较多,且细胞免疫化学法检测显示多为CD68和CDllb阳性细胞,流式细胞技术检测显示改良后的胰蛋白酶预消化加轻拍打法所收集到的CDllb阳性细胞所占细胞总数的百分比超过了92%,而传统的McCarthy方法收集的CDl1b阳性细胞纯度只有80%,并且两种纯化方法所收集的CD11b阳性细胞的纯度有显著的统计学差异(p<0.05),因此,经改良的McCarthy方法分离纯化的细胞达到了实验要求,可以作为种子细胞进行以下实验。
结论：胰蛋白酶预消化加轻拍打法可以分离纯化到纯度较高、数量较多的小胶质细胞。
目的：体内研究活化小胶质细胞在大体水平对脑梗死急性期小鼠的神经功能和梗死体积的影响。
方法：采用羧基荧光素二乙酸盐琥珀酰亚胺酯(CFDA-SE)液对小胶质细胞进行标记,并于标记后12、24和72h及7d在荧光显微镜下观察荧光信号,随后用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法检测CFDA-SE对小胶质细胞生长及增殖的影响。采用线栓栓塞大脑中动脉(MCAO)方法制做脑梗死动物模型；在观察荧光信号达到实验要求的72h后,将CFDA-SE标记的小胶质细胞经锁骨下静脉注入到MCAO小鼠体内,并分别于移植后的12、24和72h取小鼠大脑做冰冻切片以观察小胶质细胞在脑内的分布与数量。完成上述的细胞标记与示踪实验后,将66只清结级昆明小鼠随机分为四组：A组(MCAO+小胶质细胞移植,小胶质细胞移植组),B组(MCAO+MG培养基,安慰剂组),C组(MCAO组,空白对照组),D组(假手术+小胶质细胞移植,假手术组),其中A组MCAO小鼠在术后12h接受50ì1小胶质细胞悬液(1×105/μ1)；B组MCAO小鼠在术后12h接受与A组相同体积的培养基；C组小鼠在经MCAO术未接受任何处理；D组小鼠在经假手术12h后接受了与A组相同体积的小胶质细胞悬液。然后分别于小胶质细胞移植后的12、24和72h采用Zea-longa和圆筒试验对各组小鼠进行神经功能评分,随后处死小鼠后取脑,采用2%红四氮唑(TTC)法测量脑梗死的体积。
结果：细胞标记的结果显示：于CFDA-SE标记后的12h可在荧光显微镜下观察到带有很强绿色荧光信号的小胶质细胞,并且该荧光信号一直持续到第7天,说明该标记液达到了本实验的要求,另外,MTT法检测显示CFDA-SE并没有影响小胶质细胞的生长与增殖。脑组织的冰冻切片显示在移植后的12h,带有荧光信号的小胶质细胞开始在MCAO小鼠脑内出现,在移植后的72h明显增多,并且主要集中在脑梗死周围。神经功能评定显示与安慰剂B组和空白对照C组相比,MG移植A组小鼠Zea-longa评分降低,患侧前肢首次接触瓶壁的次数也明显增加,并且在统计学上有显著的差异(p<0.05)。TTC染色显示A组小鼠的梗死体积显著小于B和C组小鼠(p<0.05)。
结论：CFDA-SE标记液能够对小胶质细胞进行绿色荧光标记,并且标记的荧光信号能够维持到标记后的第7天而没有影响小胶质细胞的生长与增殖；体外移植的带有绿色荧光信号的小胶质细胞能够通过血脑屏障到达MCAO小鼠脑内,并且在移植后72h在脑梗死周围明显增多；体外移植的活化小胶质细胞改善了MCAO小鼠前肢的神经功能,减小了梗死体积。
目的：在组织及分子水平研究小胶质细胞是否影响了脑梗死急性期神经元的死亡和相关细胞因子的释放。
方法：25-35g清洁级雄性昆明小鼠264只。动物的分组及处理同第二章的实验内容。分别于小胶质细胞移植后的12h、24h和72h采用过量麻醉方法处死小鼠并取脑,然后采用HE染色法检测坏死的神经细胞,采用TUNEL染色法检测凋亡的神经细胞,采用免疫组织化学方法检测脑源性神经营养因子(BDNF)、胶质源性神经营养因子(GDNF)、肿瘤坏死因子(TNF-α)和白细胞介素-1β(IL-1β)等细胞因子及微管相关蛋白(MAP2)的表达,采用免疫荧光双染法检测了磷酸化的细胞外信号调节激酶(p-ERK1/2)在神经细胞、小胶质细胞和星型胶质细胞内的表达,另外,采用western blot方法检测了神经细胞的裂解的膜收缩蛋白(cleaved α-spectrin,神经元的骨架结构)的表达。
结果：在小胶质细胞移植后的24和72h与B和C组小鼠相比,A组小鼠的HE阳性细胞数量明显减少,而TUNEL阳性细胞、GDNF、TNF-α和IL-1β阳性细胞却无显著的增加或减少,BDNF和MAP2的阳性细胞数量显著地增加,另外,cleaved α-spectrin(分子量为145KDa)的表达水平也明显升高。免疫荧光双染显示p-ERK1/2除少量表达于小胶质细胞外,还大量地表达于神经细胞核。
结论：活化小胶质细胞抑制了神经细胞的坏死并有助于神经细胞的存活,促进了BDNF及p-ERK1/2的表达,但并没有影响神经细胞的调亡和TNF-a、IL-1β和GDNF的表达。
目的：研究活化小胶质细胞在脑梗死急性期对P-ERK1/2表达的影响及其影响的意义,为活化小胶质细胞的神经保护机制提供了理论依据。
方法：198只清洁级昆明小鼠被随机分为四组：A组(MCAO+小胶质细胞移植,小胶质细胞移植组),B组(MCAO+小胶质细胞移植+U0126,U0126抑制剂组),C组(MCAO,对照组),D组(假手术组),其中A和B组MCAO小鼠在术后12小时接受50μl小胶质细胞悬液(1×105细胞/μ1)的移植；B组小鼠在术后接受一定剂量的U0126溶液；C组小鼠在手术后未接受任何处理。在建立脑梗死或者假手术模型后,立即将U0126溶液按照30mg/kg的剂量经腹腔注入B组小鼠,以后每6小时注射1次直到实验完成,以维持一定的血药浓度。分别于小胶质细胞移植后的12、24和72h取脑,采用免疫组化方法检测BDNF阳性细胞数量,采用RT-PCR方法在基因水平检测BDNF及钙结合蛋白酶D28K(Calbindin-D28K)在各组的表达,采用western-blot方法检测了P-ERK1/2、ERK1/2、BDNF、Calbindin-D28k和cleaved-a-spectrin的表达。
结果：与B和C组小鼠相比,A组小鼠脑内的p-ERK1/2的蛋白表达水平和BDNF阳性细胞数量在移植后24和72h显著增加(P<0.05)。Rt-PCR和western-blot检测结果显示A组小鼠脑内BDNF的表达水平在移植后24和72h显著高于B和C组小鼠(P<0.05),但A组Calbindi-D28k表达的时间较晚,在移植后72h才与B和C组有显著差异(P<0.05)。另外,在移植后的72h,A组小鼠脑内a-spectrin(分子量为145KDa)裂解片段的蛋白表达水平显著低于B和C组(P<0.05),而B和C组小鼠脑内a-spectrin(分子量为145KDa)裂解片段的蛋白表达水平却无显著差异(P>0.05)。
结论：活化小胶质细胞在脑梗死急性期通过p-ERK1/2信号通路促进了神经细胞内的BDNF的表达,进而BDNF促进了Calbindin-D28k蛋白的表达,最终抑制了神经细胞的坏死。
Background
Cerebrovascular disease (CVD), served as a common disease of central neurvous system (CNS), is a major issue of public health. Cerebral infarct, also named ischemic stroke, is the disease that the death of hypoxic and ischemic brain tissue resulting from the disorder of blood supply leads to the symptom of neurological deficits. Cerebral infarct, which is the most common type of CVD and accounts for80%of the incidence, is another high mortility disease followed ischemic heart disease. Even so some patiens survives from the disease, they remains severel disabled, thus imposing a heavy burden to fomily and community. In addition, the incidence is predicted to grow with the increasing age of the population. Therefore, finding a safe and effective therapeutic method has been major purpose of medicine. At present, most studies indicated that brain injury attributed to those factors such as microglial activity under cerebral hypoxia and ischemical condition, release of proinflammar factors and excitory amino acids and generation of oxygen free radicals. However, the exact mechanisms remain unclear. MG are major immunocytes and play neurotrophic, neuroprotective and repaireing neuron cells role. Rest MG provide immuno-minitor role but the effect of activated MG in central nervous disease (CND) is still debated. Previous in vitro studies displayed that activated MG provide a neuroprotective role in CND through releasing neurotrophic factors or inhibit neurons apoptosis. Therefore, identifying the effect of activated MG after cerebral infarct would contribute to explain the pathological process of cerebral infarct and to find new therapeutic method. Therefore, it is necessary that further studying role of MG in CND in order to reveal the exact effect and mechanism of activated MG
Extracellular signal-regulated kinase1/2(ERK1/2), one of the best-characterized members of the mitogen-activated protein kinase (MAPK) family, mediates a range of activity from metabolism, motility, and inflammation to cell death and survival. It is phosphorylated and activated through a three-tiered MEK mode via cell surface receptors stimulated by growth factors or cytokines. Most phosphorylated ERK1/2translocates to cell nucleus and induces genic transduction and expression and is closely associated with cell proliferation and differentiation. It is well documented that phosphor-ERK1/2mainly expresses in neurons of cortex in peri-infarct sites after acute cerebra ischemia. It is also reported that ERK1/2located in MG is phosphorylated in response to acute cerebral infarct and phosphor-ERK1/2promotes MG to synthesize and secrete brain-derived neurotrophic factor (BDNF).
BDFN is a member of neurotrophic factor family and generally locals in CNS, sensory neurons and the neurons of spinal cord motor. Recently, numerous studies from world have confirmed that BDNF not only play a critical role in neuron surviva, differentiation and sustaining normal physiological function but protecting neurons against injury. Meanwhile, it also promotes neuron regeneration. However, more views supported that the neuroprotective roles of BDNF are due to inhibiting cytotoxity of excitory amino acids and stabling Ca2+concentration of cytoplasm. Some studies have showed that BDNF prevented Ca2+from entering into cell and secreting from cytoplasm. The BDNF treatment increases the number of hippocampus neuron with calcium binding protein (CBP). The neurons with CBP show more significant ability in resisting glumatic acid cytotoxity and reducing Ca2+concentration in cytoplasm than neurons not with CBP. Therefore, BDNF could stable Ca2+concentration in cytoplasm through CBP role.
Calbindin-D28k is a major member of CBP family. Recently, many experts have done numerous in vivo and in vitro studies to explore the effect of calbindin-D28k. although calbindin-D28k locates in neurons, it is not linked with neurotransmitter at all. It indicated that calbindin-D28k role in mediating Ca2+concentration in brain is associated with the some phosiological repense of neurons with CBP. Calbindin-D28k plays a role in synaps linking and maybe prevents neurons subjecting to injury due to excessive Ca2+concentration-depended on voltage difference. Calbindin-D28k stabilizes Ca2+concentration in cytoplasm through stably binding Ca2+and buffering and transporting Ca2+to arrest over Ca2+accumulation. Calbindin-D28k can activate Ca2+/Mg2+/-ATP enzyme activation and arrest Ca2+excessive accumulation in cytoplasm. It is a critical factor in sustaining Ca2+balance that calbindin-D28k bufferes and transports excessive Ca2+in neuronal plasm. Many studies have revealed that calbindin-D28k can remarkly reduce neurons injury induced by hypoxia and ischemia. In addition to this, the neuroprotective role of Calbindin-D28k maybe be associated with inhibiting calpain activation. Excessive elevation of cytosolic Ca2+levels after acute cerebral infarct activates calpain. Activated calpain can cleave a-spectrin into two protein fragments as molecular weight of145and150KDa and neurons necrosis is increased.
Since MG play a critical role in ischemic cerebral injury and the effect and molecular mechanisms of atctivated MG in response to cerebral infarct especially actue cerebral infarct remain unclear, it is necessary to further study the role of activated microglia under in vivo condition after acute cerebral ischemia. The present study maybe provide new method and potential target for treating acute cerebral infarct.
Objective:To provide transplanting microglia for detecting the effect of activated microglia after acute cerebral ischemia.
Methods:Mixed glial cells were obtained from cortex of SPF Kunming mouse embryo for14days. Traditional McCarthy and modulated McCarthy method i.e trypsin predigesting combinated with slight tapping were employed to extract and purify MG Then immunocytochemistry was used to detect the antigenic markers of cell and flow cytometry was employed to determined cultured cell purity. Finally, purity cell was cultured or subcultured.
Results:Primary cultured results showed that more cells were obtained by modified McCarthy method than traditional McCarthy method. Immunocyto chemistry determining showed that the surface antigen of most puriftied cells was CD68and CDllb. the results of flow cytometry technique showed the number of CD11b positive cells was over92%of total cells number by modilated method but80%by McCarthy and there was significant difference between modilated and traditional McCarthy methods in the number of CDllb positive cells (p<0.05). Therefore, these cells by isolating and puritfy were microglia and can be employed to the studies of microglial function.
Conclusions:Modified McCarthy method improves microglial yield and purity.
Objective:To determine effect of activated microglia on neurological function and infarct volume of mouse after acute cerebral ischemia in vitro.
Methods:First, MG were marked by CFDA-SE with green fluorescence and the signal intension of fluorescence was observed under fluorescence microscope at12,24and72h as well as7day after marking. In addition, MTT was employed to detect the effect of CFDA-SE on the growth and proliferation of MG. Secondly, Once the signal intension reached the experiment criterion, MG marked by CFDA-SE were injected into permenant middle cerebral artery occlusion (pMCAO) mice body through subclavian vein, and the brains were disassociated at12,24and72h. The disassociated brains were sliced by frost slice techniques and the number of MG with fluorescence was observed under fluorescence. Finally,66mice were divided randomly into4groups:A group (MCAO+MG transplanting), B group (MCAO+MG medium), C group (MCAO group) and D group (sham operation+MG transplanting). Triphenyltetrazolium chloride (TTC) was employed to detect infarct volume and Zea-longa and cylinder test were used to evaluate neurological deficits at12,24and72h after transplanting.
Results:The results derived from cell marking showed that the flueroscence intension of marked cells was significant and unchanged at at12,24and72h after treatment. However, the signal intension of attached cells was partially attenuated at7day after treatment. MTT test revealed that CFDA-SE did not affect the growth and proliferation of microglia. The results derived from cell tracing showed that few cells marked with flueroscence was observed at12h after transplanting and the number of marked cells was increased at24and further increased at72h after treatment. In addition, the marked cells mainly scattered in peri-infarct site at72h after treatment. Compared with B and C groups, mice in A group had smaller infarct volume, lower Zea-longa score and more frequencies touching bottle wall with hemi-paretic forepaws at24and72h after transplanting (P<0.05).
Conclusions:CFDA-SE marking liquid is a good liquid marking MG and dose not affect MG growing and proliferation. CFDA-SE sustains intensive flueroscene signal for7days and it has met the criterion of present experiment. Primary cultured MG can enter the peri-infarct site of MCAO mice through blood brain barrier and the number of MG in brain is increased at72h after transplanting. Activated microglia play a neuroprotective role generally after acute cerebral ischemia.
Objective:To detect whether activated microglia influence neuronal death and releasing of cytokines after acute cerebral ischemia in tissue and molecular levels.
Methods:Two hundred sixty four adult male SPF kunming mouse weighing25-35g were used in present section. The animal grouping and treating were similar with Part Ⅱ. Nectrotic and apoptosic neuron cells, survival neurons and proinflammar and neurotrophic factors, such as TNF-a, INL-1β,GDNF and BDNF, in brain of mice in4groups were determined by HE, TUNEL, immunohistochemistry at12,24and72h after transplanting. Duable flueronscence staining was used to detect phosphorylated ERK1/2expression on MG, astrocytes and neurons. Western blot was used to test the expression levels of cleaved a-spectrin.
Results:Compared with B and C groups, mice in A group showed less HE positive cells but similar number in TUNEL positive cells in peri-infarct at24and72h after transplanting (P<0.05). There were no difference among the three groups in TNF-a, IL-1β and GDNF positive cells number at24and72h after treatment (P>0.05). However, the number of BDNF positive cells was remarkly increased compared with B and C groups (P<0.05). Duable flueroscene staining showed that pERKl/2mainly located in neuronal nuclear in the brain of MCAO or sham-operation mice. Compared with B, C and D group, the number of pERK1/2positive cells was remarkly increased at24and72h after transplantion. In adddition, there was no difference between B and C groups in the number of pERKl/2positive cells (P>0.05).
Conclusions:Activated microglia inhibit neuronal cells necrosis and promote BDNF and p-ERK1/2expressions after acute cerebral ischemia.
Objective:To determine the effect of activated microglia on the expression of p-ERK1/2and significance of the effect after acute cerebral ischemia, and to provide theory evidence.
Methods:Two hundred and sixteen mice were divided randomly into4groups: A group (MCAO+MG transplanting), B group (MCAO+MG transplanting+U0126, inhibitor group), C group (MCAO group) and D group (sham operation). Western blot was used to test the expression levels of pERKl/2, BDNF, calbindin-D28k and cleaved a-spectrin at12,24and72h after acute cerebral infarct. Immunohistochemistry and RT-PCR were used to detect the number of BDNF positive cells and the expression level of BNDF and calbindin-D28k mRNA respectively at the three time points.
Results:Compared with B and C groups, expression levels of pERK1/2protein and the number of BDNF positive cells of mice in A group were increased at24and72h after transplanting (P<0.05). The expression levels of BDNF mRNA and protein were increased at24and72h after transplanting and the increase in expression level of calbindin-D28k protein was at72h (P<0.05). In addition, the expression level of cleaved a-spetrin protein in A group was remarkably increased compared with B and C group at72h after treatment (P<0.05). However, there was no difference between B and C groups in the expression of pERKl/2, BDNF, calbindin-D28k and cleaved a-spectrin protein and mRNA expressions of BDNF and calbindin-D28k (P>0.05).
Conclusions:Activated microglia up-regulate expression of BDNF through ERK1/2signal pathway. BDNF inhibit neuronal necrosis through promoting expression of Calbindin-D28K after acute cerebral ischemia.
引文
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