Investigation Into Mechanism Of APOE Subtype-Specific Effect On Secondary Injury Of Neuron And Glial Cell

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 Ca²⁺ influx and may play an important role in Ca²⁺ 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 genotypeAPOE 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 NSCsIsolation, 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 injuryThe 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 injuryThis 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 Ca²⁺ overload, which may be one of the mechanisms APOEε4 can adversely affecting the outcome of TBI.