Contributions Of Ngb And HSP60in The Pathologic Process Of Contusion And Laceration Of Brain

Research background Contusion and laceration of brain is a frequent severe neurosurgical disease. It is one of the principal diseases threatening human life. Though the medical science is more advanced than before, Mutilation and lethality of contusion and laceration of brain is still higher and higher now and there is no extremely effective therapeutic method on these days. So it is very important to follow the pathogenesis of this kind of disease and study the causative agent for the clinical practice. Ngb (Neuroglobin) is a new globin that is situated mainly in neurons and be an endogenous neuroprotector. Being similar to Hgb (Hemoglobin) and Mgb (Myoglobin), Ngb can reversibly combine oxygen. Lots of new researches indicate that Ngb plays a role of neuroprotection in the disease of brain ischemia-hypoxia. Hsp60(Heat Shock Protein60) is situated mainly in mitochondria in cells and help opypeptides or proteins to dislocate, fold and assemble. HSP60also play an important role in the control of the stress. When the bad stimulations come, the expression of HSP60increases immediately, and maintains space conformation of proteins in cells. The vital movement and survival ability of cells is protected accordingly. To study Ngb and HSP60in the pathologic processes of contusion and laceration of brain, we have maken some interesting experiments recently.There are three parts of my topic:Chapter one:The expression of different proteome in the brain tissue and blood of the patients with contusion and laceration of brainChapter two:The expression of Ngb and HSP60in in the brain tissue of the patients with contusion and laceration of brainChpter three:The expression of of Ngb and HSP60in in the serum of the patients with contusion and laceration of brain Chapter1. The expression of comparative proteome of brain tissue and leukocyte in patients with contusion and laceration of brainObjective:To establish two-dimensional electrophoresis profiles with high resolution and reproducibility from brain tissue and leucocyte in blood of the patients with contusion and laceration of brain. And to analyse the different expression proteins, investigate the function and effect of them.Methods:Brain tissue and blood was collected from ten patients with contusion and laceration of brain, eight patients with intraventricular neoplasms who had to be taken operation and ten normal volunteers. The leukocytes were separated from blood by lydroxypropylmethyl cellulose. The proteins in brain tissue and leukocytes were extracted by two-dimensional gel electrophoresis. The image analysis were done using the PDQuest7.0software. The differential protein spots were detected and analyzed with Applied Biosystems Voyager System4307MALDI-TOF Mass Spectrometer and bioinformatical skills.Results:The proteins were acquired from brain tissue and leukocytes separately and analyzed with the PDQuest7.0software. In the part of brain tissue, there were764±28proteins in the normal group while753±19proteins in the contusion and laceration of brain group. The stained2-DE gels were scanned by Imagescanner. The image analysis were done using the PDQuest7.0software. To compare with the normal group, the expressions of proteins in the contusion and laceration of brain group were different,15spots of proteins were up-regulated while16spots were down-regulated. Part of the spots were detected with the mass spectrum and Protein Databases and seven proteins came out:Trx (Thioredoxin), UCH-L1(Ubiquitin carboxy-terminal hydrolase isozyme L1), Ngb (Neuroglobin), NSFL1cofactor p47, Microtubulin alpha chain, Dihydropyrimidinase-related protein2, Synaptosomal associated25K protein. The proteins were acquired from brain tissue and leukocytes separately and analyzed with the PDQuest7.0software. In the part of leukocyte, there were572±18proteins in the normal group while518±16proteins in the contusion and laceration of brain group. The stained2-DE gels were scanned by Imagescanner. The image analysis were done using the PDQuest7.0software. To compare with the normal group, the expressions of proteins in the contusion and laceration of brain group were different,21spots of proteins were up-regulated while17spots were down-regulated. Part of the spots were detected with the mass spectrum and Protein Databases and six proteins came out:Calreticulin precursor, Hsp60(60kDa Heat shock protein), Aldehyde dehydrogenase Calcium-dependent protein kinase, Fatty acyl-coa dehydrogenase and Glutamate dehydrogenase precursor.Conclusion:1. We have established the expression maps of brain tissue and leukocytes in the patient with contusion and laceration of brain and the normal group with two-dimensional gel electrophoresis.2.The differences of the two-dimensional expression maps between the contusion and laceration of brain group and normal group were found after the image analysis of the gels.3. The proteins with differential expression were detected and analyzed with mass spectrometer and Protein Databases. They could take part in the pathologic processes of contusion and laceration of brain by oxidation-reduction reaction, division of cell and access of protein degradation, signal transduction. Among these proteins, Ngb and Hsp60may play very important roles in the pathologic processes.Chapter2:The expressions of Ngb and HSP60in the brain tissues of the patients with contusion and laceration of brainObjective:To detect the expression of Ngb and HSP60in the brain tissue of the patients with contusion and laceration of brain and the normal group with immunohistochemical skills and find the differences between two groups, then to evaluate contributions of Ngb and HSP60in the pathologic process of contusion and laceration of brain.Methods:Using streptavidin-perosidase (SP) method, according to the directions of the kit supplied from Zhongshan Goldenbridge Biotechnology CO., LTD Beijing. Brain tissues obtained from ten patients with contusion and laceration of brain, all the lesions were at the right lobus frontalis. The normal brain tissues obtained from lobus frontalis of ten patients with brain ventricular neoplasms who had to take operations. Then got slices, deparaffinage and hydration, rinse, wash with distilled water, to repair by microwaves, incubate with first and second antibodys, observe with light microscope, stain with hematoxylin, mount with gum, observe the staining with light microscope and take pictures.Results:1. Expressions of Ngb were in plasm of cells, the dilute brow object revealed the positive expressions. The expressions of Ngb were weakly positive in normal brain tissues, while it was positive in brain tissues with contusion and laceration and it was hadro-positive in the tissues closed to brain tissues with contusion and laceration. Statistical significant difference could observe between two groups (P<0.05).2. Expressions of HSP60were in plasm of cells, the dilute brow object revealed the positive expressions. The expressions of HSP60were positive in normal brain tissues, while it was positive in brain tissues with contusion and laceration and it was hadro-positive in the tissues closed to brain tissues with contusion and laceration. Statistical significant difference could be observed between two groups (P<0.05).Conclusion:The expressions of Ngb and HSP60were hadro-positive in the tissues closed to brain tissues with contusion and laceration. Ngb and HSP60may play important roles in the pathologic process of contusion and laceration of brain.Chpter three:The expressions of Ngb and HSP60in the serum of the patients with contusion and laceration of brainObjective:To detect the expression of Ngb and HSP60in serum of the patients with contusion and laceration of brain and the normal group with Western blotting and find the differences between two groups, then to evaluate contributions of Ngb and HSP60in the pathologic process of contusion and laceration of brain.Methods:There were two groups, one was make up of ten patients with contusion and laceration of brain who had not taken operation, another was make up of ten volunteers. The sample of serum was diluted, concentration of proteins were detected, then polyacrylamide gel electrophoresis was taken, transfer to PVDF membranes, incubate with first and second antibodys, luminescence and developing with ECL reagent. Get image after scan, software of image analysis, to detect gray scale, then analize the special protein.Results:1. Expressions of Ngb were low in normal serum; Expressions of Ngb up-regulated three hours after contusion and laceration, peak of the expression appeared at12hours after contusion and laceration and the expressions down-regulated then, Ngb became normal at72hours after contusion and laceration. Statistical significant difference could be observed between the two groups at6h and12h after contusion and laceration (P<0.05).2. Expressions of HSP60were low in normal serum; Expressions of Ngb up-regulated three hours after contusion and laceration, peak of the expression appeared at72hours after contusion and laceration. Statistical significant difference could be observed between the two groups at24h,48h and72h after contusion and laceration (P<0.05).Conclusion:The expressions of Ngb and HSP60were hadro-positive in the serum of patients with contusion and laceration, Statistical significant difference could be observed between normal group and patients. Ngb and HSP60may play important roles in the pathologic process of contusion and laceration of brain.