The Neural Cells Were Protected By Neuroglobin (NGB) In Cerebral Ischemia Of Rat Brain

Ischemic cerebrovascular disease is a common disease of older persons, and it is also a major disease which leads to death and disability for the elderly. Because its pathogenesis is very complex and its etiology is unknown, there hasn't been a drug which can treat it thoroughly or reverse its course for the time. Oxygen is the necessary factor which sustains the metabolism and subsistence of the organism. Up to now, we have found four kinds of globulins in vertebrates. They are hemoglobin (HGB) which saves and transports oxygen, myoglobin (MGB) which helps oxygen enter into mitochondrial, cytoglobin which conduces to the diffusion of oxygen and the clearance of cytotoxicity nitrogen monoxidum as a buffer and neuroglobin (NGB) which we found recently. Neuroglobin (NGB) is an invertebrate monomer globin. It has been proved to store, transfer oxygen and protect neural cells, which can increase oxygen diffusion into the mitochondria, protect neural cell. This topic is to study whether pCDNA3.1/ Ngb recombinant plasmid can protect neural cells and its effect factors related.1 Cloning of rat neuroglobin gene and construction of itsexpression vector 1.1 Cloning of rat neuroglobin gene The total RNA was extracted from Wistar rat brain and the full length cDNA encoding NGB was obtained by RT-PCR.After the sequence was confirmed by sequencing and BLAST. Results The NGB gene was cloned, four bases were mutated.Conclusion There is expression of NGB in Wistar brain and NGB can be used in further study.1.2 Construction of rat neuroglobin gene eukaryotic expression vectorThe total RNA was extracted from Wistar rat brain and the full length cDNA encoding NGB was obtained by RT-PCR. After the sequence was confirmed by sequencing and BLAST, it was inserted in the eukaryotic expression vector pCDNA3.1 (+), then the sequence and reading frame were confirmed by two restriction endonucleases and sequencing. Results The NGB gene was cloned, four bases were mutated and its eukaryotic expression vector was constructed. Conclusion There was expression of NGB in Wistar brain. The NGB gene was successfully cloned and inserted in eukaryotic expression vector.1.3 Prokaryotic expression and polyclonal antibody preparation of rat neuroglobin gene The total RNA was extracted from Wistar rat brain and the full length cDNA encoding NGB was obtained by RT-PCR. After the sequence was confirmed by sequencing, it was inserted in the prokaryotic expression vector pET-28(+), then the sequence and reading frame were confirmed by two restriction endonucleases and sequencing. And it was expressed in E.coli BL21. The protein was used to immune the rabbit for preparing polyclonal antibody after it was identified and purified. The antibody titer was tested by ELISA method. Results The titer of polyclonal antibody is 100 000. Conclusion The polyclonal antibody of NGB was prepared and can be used in further study.2 Establishment of rat focal cerebral ischemia model40 Wistar male rats were randomized into the conventional suture group and the modified suture group,20 per each group, and were used to induce focal cerebral ischemia by MCAO according to Longa's suture method.The effects of the application of modified suture on the total operation time, the common carotid artery blood occlusion time during the course of operation, the probability that suture entered the pterygopalatine artery (PPA) and the success rates were observed. And the stability and the reliability of the model were evaluated by neurological deficit scores, magnetic resonance image, triphenyltetrazolium chloride (TTC) staining in all rats.Results The total operation time and the common carotid artery blood occlusion time in the modified suture group were obviously shorter than those in the conventional suture group. And the application of modified suture in the model cut down the probability that the suture enter PPA, and increased in the success rates, the stability and the reliability of the model. Conclusion The application of modified suture could improve the success rates, the stability and the reliability of the rat focal cerebral ischemia model induced by MCAO.3 The neural cells were protected by recombinant plasmid pCDNA3. 1(+)/Ngb in cerebral ischemia of rat brain54 male Wistar rats were randomly divided into three groups:normal saline(NS) control group,plasmid control group,and recombinant neuroglobin group (pCDNA3.1(+)/Ngb).NS,plasmid pCDNA3.1(+) and recombinant plasmid pCDNA3.1(+)/Ngb were respectively injected into two sites in the rat cerebra1 cortex 24 hours before induction of neocortical focal ischemia by occlusion of the right middle cerebral artery for 24 hours. The condition of local ischemic damage, expression of bcl-2 and the apoptosis in neural cells were confirmed by staining with 2% 2.3.5-triphenyltetrazolium chloride , in site cell apoptosis detection, indirect immunofluorescent staining and western blot ,respectively.Results The extent of cerebral infarction tissue and the apoptosis cells in the pCDNA3.1(+)/Ngb group were significantly reduced than those in other control groups (P <0.01).The number of expression bcl-2 cells in pCDNA3.1(+)/Ngb group was much more than that in other control groups (P<0.01).The relative expression level of bcl-2 protein was about up 40%～50% by western blot analysis. Conclusion The neural cells were protected by recombinant plasmid pCDNA3.1(+)/Ngb in focal cerebral ischemia, because apoptosis of neural cells was inhibited through being increased the expression of bcl-2 gene.