摘要
创伤性脑损伤(traumatic brain injury, TBI)是神经外科最常见的一种疾病,其死亡率、致残率居各类创伤之首。脑伤后以细胞凋亡为主要特征的继发性脑损伤是影响伤情转归的主要因素。载脂蛋白E基因(apolipoprotein E, APOE)主要有ε2、ε3、ε4三个等位基因,我们前期工作和国外研究显示,携带ε4的患者对脑损伤的耐受性降低,更易出现伤情加重及不良预后。但是,APOE影响脑损伤病情转归的机制尚未阐明。Ca~(2+)超载是细胞死亡的“最终共同通路”,Ca~(2+)超载实质是细胞内外离子失衡的结果。K~+是维持细胞内离子环境最主要的阳离子,目前认为钾通道也是细胞凋亡的重要环节之一。本课题以APOE亚型特异性与延迟整流钾通道的关系作为切入点,构建人源性ε2、ε3、ε4的真核表达载体,分别转染APOE敲除鼠胚神经干细胞(neural stem cells, NSCs),筛选稳定表达细胞株,建立神经元/胶质细胞共培养划痕损伤模型,采用流式细胞技术及膜片钳技术分析伤后APOE亚型特异性与细胞早期凋亡、延迟整流钾通道的关系,探讨APOE影响继发性神经细胞损伤的作用机制。
一、人源性APOE基因真核细胞表达载体的构建和鉴定
人源胎脑组织中提取Total RNA后,设计引物调取APOEε3基因CDS区域,克隆至pMD19-T simple载体中并测序验证,采用定点突变技术,得到APOEε2及APOEε4的重组突变体载体。EcoR I/BamH I双酶切切下编码APOE的片段,插入真核表达载体pEGFP-N1。对重组质粒进行双酶切、PCR和测序验证后,采用阳离子脂质体Lipofectamine 2000将重组质粒转染至真核细胞(293-T细胞)中,用荧光显微镜观察基因在239-T细胞中的表达情况。结果:双酶切、PCR和质粒测序结果证明APOE各等位基因正确地克隆到真核表达载体pEGFP-N1中,并能在293-T真核细胞中进行融合表达。结论:成功构建了人源性pEGFP-N1-APOE各亚型的真核表达载体,并成功转染真核细胞,为下一步实验中各重组质粒转染APOE敲除鼠的NSCs奠定了基础。
二、稳定表达pEGFP-N1-APOE的神经干细胞克隆的建立和鉴定
采用APOE敲除鼠(E15d)的胎鼠脑组织为细胞来源,常规分离,培养和鉴定所得到的NSCs。将前期构建好的人源性APOE各等位基因重组质粒以阳离子脂质体Lipofectamine 2000转染至NSCs中,在含有200μg/ml G418培养液中培养14d,以筛选稳定表达pEGFP-N1-APOE的NSCs克隆。将稳定表达的细胞克隆进行单细胞克隆培养以纯化稳定表达人源性APOE各等位基因的NSCs。扩增稳定表达目的基因的NSCs,用RT-PCR、Western Blot、激光共聚焦显微镜检测目的基因在转基因NSCs中的表达情况。结果:成功将前期构建的人源性APOE各重组质粒转入APOE敲除鼠NSCs,经检测证明稳定表达EGFP的NSCs可表达人APOE各等位基因。结论:采用阳离子脂质体法可有效地将APOE各重组质粒转染到NSCs,经过G418筛选后可以获得稳定表达pEGFP-N1-APOE的NSCs克隆。
三、APOE亚型特异性对神经细胞伤后早期凋亡的影响
本实验采用稳定表达人APOE各等位基因的APOE敲除鼠NSCs,优化诱导分化条件,构建神经元/胶质细胞共培养体系。控制条件,利用微量移液器枪头切割培养的神经元、胶质细胞,造成神经细胞机械性损伤,构建细胞划痕损伤模型。参考文献及预实验结果设置时间点(6h、12h、24h、48h),通过Annexin V/PI联合流式细胞技术检测损伤后各组细胞早期凋亡率,分析人APOE各等位基因影响继发性神经细胞损伤的差异。结果:成功构建携带人源性APOE各等位基因的细胞划痕损伤模型;伤后各时间点均能检测到早期凋亡细胞,24h时间点各组细胞早期凋亡率较6h、12h明显增高,有显著性差异(P<0.05),且人APOEε4组及鼠APOE(?)组较其余组早期凋亡率明显增高,有显著性差异(P<0.05)。结论:转染人APOEε4的细胞早期凋亡率高于其他组,从细胞水平为APOE多态性影响TBI病情及预后的临床研究结果提供了实验依据。
四、APOE亚型特异性影响神经细胞早期凋亡的机制探讨
以APOE亚型特异性与延迟整流钾通道相关性作为切入点,采用膜片钳技术检测各实验组延迟整流钾电流(I_D)的差异。选择边缘清晰、大小相似的神经元样细胞作为实验对象,记录致伤前后I_D情况,并进行组间比较,进一步探讨APOE影响神经细胞早期凋亡的病理机制。结果:(1)依据电生理特性证实各细胞组所记录到的外向电流为I_D。且发现未受伤状态下各组神经细胞的I_D无明显差异(p>0.05)。(2)在机械损伤后,各组神经细胞的I_D均明显增加,但是人APOEε4组与鼠APOE(-)组抑制神经细胞I_D幅度增加的作用较其余组明显(p<0.05)。结论:APOEε4对伤后神经细胞的I_D抑制作用更明显,造成APOEε4细胞内Ca~(2+)超载较APOEε2和APOEε3细胞严重,这可能是其影响TBI后病情转归及预后的机制之一。
Traumatic brain injury (TBI) is an important global public health problem and a leading cause of morbidity and mortality worldwide in the neurosurgery field. Secondary brain injury featuring apoptosis following primary damage is one of the major factors which affected the outcome of TBI. In humans, apoE is the polymorphic protein, with three common isoforms (apoE2, E3, E4), encoded by three alleles (ε2,ε3,ε4) of a single gene on chromosome 19q13.2. Overseas and our previous studies suggests that the patients with APOEε4 predispose to clinical deterioration in acute and chronic phase after TBI and this may contribute to the poor outcome after head injury. But the mechanism involving why the APOEε4 can adversely affect the outcome is unclear. It has been suggested that calcium-mediated mechanisms were the“final common pathway”leading to cell death following CNS injury. K~+ efflux can affect Ca~(2+) influx and may play an important role in Ca~(2+) overload. The decrease of intracellular potassium concentration ([K~+]_i) is one of the features of cell apoptosis. This study focuses on the relationship between the APOE genotype and delayed rectification potassium channel to explore the mechanism. Construct of eucaryotic expression vector carrying each APOE genotype (ε2,ε3,ε4) was performed. The recombinant plasmid was transfected into neural stem cells (NSCs) from APOE knock-out mice with Lipofectamine 2000. NSCs stablely expressing APOE were purified by single cell cloning culture. An in vitro model of mechanical injured neuron/glial co-culture system was established. Flow cytometry and patch clamp technique were used to analyze the relationship among APOE genotype, early cell apoptosis and delayed rectification potassium channel to elucidate the mechanism invoving APOE genotype affecting secondary neuron cell damage.
1. Construct and identification of eucaryotic expression vector carrying human APOE genotype
APOE was amplified from human embryo pallium of fetus by PCR, inserted the products of APOE PCR products into pMD19-T simple. Sequence analysis was performed to identify the correctness of APOEε3. Site-directed mutagenesis was used to obtain the cDNA encoding apoE2 and apoE4 isforms. The sections of APOE gene (ε2,ε3,ε4) were obtained by the restriction digested with EcoR I and BamH I and inserted into pEGFP-N1 respectively. Recombinant plasmid was identified by enzyme digestion, PCR and sequence analysis. Recombinant plasmid was transfected into 293-T cells with Lipofectamine 2000 and the expression of the plasmid was detected by fluorescence microscope. Results: Results of enzyme digestion, PCR and sequence analysis of recombinant plasmid demonstrated that APOE gene (ε2,ε3,ε4) were correctly inserted into eucaryotic expression vector pEGFP-N1. Recombinant plasmids were expressed successfully in 293-T cells transfected. Conclusion: A green fluorescent protein reporter gene vector containing human APOE genotype is successfully constructed, which providing an important and convenient tool to transfect NSCs of APOE knock-out mice.
2. Stable transfection and identification of pEGFP-N1-APOE into NSCs
Isolation, cultivation and purification of the primary NSCs from rat embryo pallium of APOE knock-out mice were performed. Recombinant plasmid was transfected into NSCs with Lipofectamine 2000. NSCs were selectively cultured for 14 days in the medium containing 200μg/mL G418. NSCs stablely expressing pEGFP-N1-APOE were purified by single cell cloning culture. After NSCs stablely expressing EGFP gene and APOE were amplified, expression of target gene in amplified NSCs was analyzed by RT-PCR, Western Blot and laser confocal microscope. Results: Plasmid was transfected successfully into NSCs by Lipofectamine 2000. NSCs were able to express EGFP after transfection. Some clones stablely expressing EGFP were formed 14d after selective culture. Purified clones were obtained through single cell cloning culuture. RT-PCR, Western Blot and laser confocal microscope analysis demonstrated human APOE gene (ε2,ε3,ε4) were expressed in the NSCs. Conclusion: pEGFP-N1-APOE is transfected successfully into NSCs by Lipofectamine 2000 and clones stablely expressing target gene are obtained.
3. The subtype-specific effects of APOE on early apoptosis of neurons and glial cells after injury
The 2nd generation NSCs carrying human APOE isoform were inoculated into 6 shadow mask, and differentiated after 2d adherent culture using optimized culture condition to establish the neuron/glial co-culture system. Mechanical injury was produced with a plastic tip fixed on the special device crossing onto the cultured cells. The time point for detecting apoptosis at 6h, 12h, 24h and 48h after injury was determined according to the references and our pre-experiment. Annexin V/PI flow cytometry was used to analyze the relationship between APOE genotype and early cell apoptosis. Results: The mechanical injury cell model was established successfully on neuronal/glial co-culture system. Early cell apoptosis after injury were identified in all cell groups with or without APOE genotype. Early cell apoptosis rate at 24h were higher than 6h and 12h (p<0.05) in each group. APOEε4 group and rat APOE(-) group showed severe early cell apoptosis at 24h which were statistically different in early cell apoptosis rate from another groups ( rat APOE(+) group, human APOEε2 group and human APOEε3 group) (p<0.05). Conclusion: Our results provide experimental evidence on cellular basis for the clinical findings that the patients with APOEε4 predispose to clinical deterioration in acute phase after TBI.
4. The investigation into mechanism of the subtype-specific effects of APOE on early cell apoptosis after injury
This study focuses on the relationship between the APOE genotype and delayed rectification K~+ current. The neuronal-like cells with clear edges and similar size were chosen to record current. Patch clamp technique was used to analyze the changes between APOE genotype and delayed rectification K~+ current (I_D) following mechanical injury to elucidate the mechanism invoving APOE genotype affecting secondary neuron cell damage. Results: (1) Delayed rectifying K~+ current was identified and recorded in all cell groups with or without APOE genotype and no significant differences in I_D among the groups were found before injury. (2) The increases of I_D were observed in all groups after injury, but the amplitude of increase of I_D in human APOEε4 and rat APOE(-) were significantly lower comapared repectively with any other group (p<0.05). Conclusion: The finding suggests that APOEε4 can exsert inhibition effect on delayed rectifying K~+ current after injury resulting in reduced K~+ efflux and aggravating Ca~(2+) overload, which may be one of the mechanisms APOEε4 can adversely affecting the outcome of TBI.
引文
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