The Investigation Of Key Molecules For Promoting Fracture Healing During Traumatic Brain Injury Using Proteomic And Metabonomic Methods

Clinical evidence suggests an association between traumatic brain injury (TBI)and enhanced osteogenesis. Clinical and observational studies have shown that thefractured long bones heal more quickly and make more callus in TBI patientscompared to those without TBI. Increasing incidences from patients with fracturesdemonstrated that the head injury is closely associated with heterotopic ossification(HO), rapid bone union, hypertrophic callus formation. Recent findings found thatboth serum and cerebrospinal fluid (CSF) from TBI patients promoted theproliferation of fibroblasts, mesenchymal stem cells and progenitor osteoblasts,thereby demonstrated their osteoinductive potentials. The associations between theosteoinductive factors and fracture healing in TBI patients have already beendemonstrated and these osteoinductive factors mostly come from serum. Thus, thesefindings reinforce the hypothesis that bone formation may be regulated by serumfactors through the change of concentration, which is caused by increased leakagefrom brain into blood because of blood-brain barrier (BBB) dysfunction or the resultof systemic response to TBI, and subsequently act peripherally on target tissues.Many serum factors are associated with this event, such as growth hormone,transforming growth factor-β, nerve growth factor, insulin-like growth factor II,platelet-derived growth factor, interleukin (IL)1and IL6. To date, although there is aconsensus about the enhanced fracture healing in TBI patients, the exactpathophysiological mechanism for this phenomenon remains controversial. Previousstudies focused on few proteins but were short of the systematic study on this topicusing―-omics‖approaches.The aim of this study was to investigate different molecules in TBI rat serum by ―-omics‖approach and the associations between different factors and events. Thenovel findings may provide new important evidences and clues for deeplyunderstanding the event and identifying the potential drug targets for fracturetreatment in clinics.This dissertation consists of5parts and all contents are summarized as follows:In the first chapter, we aimed to investigate the association between TBI andfracture healing. Firstly, animal models were generated according to the repeatableand reliable protocol. Secondly, the histomorphological and radiological observationsshowed that the enhanced fracture healing is associated with high bone mineraldensity, high volume of bone trabeculae and large number of osteoblasts in TBI ratswith long-bone fracture at the time point of3weeks, which is compared to the ratswith an isolated long-bone fracture. Thirdly, the serum from TBI rats promoted invitro proliferation of MC3T3-E1cells and enhanced BGLAP expression.Consequently, this finding suggests that the serum factors may relate to the formationand mineralization processes of bone metabolism through regulating the osteoblastproliferation and BGLAP expression. The results from animal model studies may laythe foundation for screening different factors.In the second chapter, the aim of this study was to compare the expression profileof Hp in TBI rats with that in normal rats using comparative proteome analysis(two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS)). Proteindifferences in serum samples from normal and TBI rats at different times wereevaluated by high resolution separation and high throughput identification techniques.Our results indicate that serum Hp was upregulated at24hours after TBI. Next, Hp isupregulated in liver probably by elevated pro-inflammatory cytokines (IL6) in TBIblood. By bioinformatics analysis, we found that Hp and IL6were relevant to theregulation of inflammatory reaction. Therefore, these findings suggest that they mayplay important roles in the inflammation stage of fracture healing.In the third chapter, gas chromatography/mass spectrometer (GC/MS) coupledwith multivariate statistical analysis was used to identify the changes of global serummetabolites after TBI. Complimentary to other profiling technologies like proteomicsand genomics, metabonomics provides a fingerprint of small molecules in livingorganisms, and a research method for understanding the associations betweenmetabolites and pathophysiological changes. The objects of research mainly consist of small molecules with molecular weight less than1000. We found the arachidonic acid(AA) was significantly enhanced in serum metabolites in TBI subjects, while otherdifferent metabolites were reduced. Furthermore, the results of cellular and molecularstudies showed that AA was able to promote in vitro proliferation of MC3T3-E1cellsand enhance BGLAP expression in MC3T3-E1cells. The previous study reported thatthe speed of fracture healing may be determined by the processes of inflammation,soft callus and hard callus; while the strength of repair is related to the mechanicallydriven balance between bone specific anabolism and catabolism during boneremodeling. Consequently, we propose that AA may regulate soft and hard callusformations by accelerating the maturation of osteoblasts in the fracture healing event.In the fourth chapter, the work focused on the differentially expressed peptidesand microRNA (miRNA) profiles using LC-LTQ and miRNA array. Increasingevidences show that small peptides and miRNA perform essential functions in liveorganisms and take part in the pathophysiological processes at several levels. Bypetidomic and miRNA analyses, different peptides and miRNAs were found in serumsamples from normal and TBI rats. This finding was not only good complement toproteomic and metabonomic studies, but also new clues for exploring andunderstanding the relation between TBI and accelerated fracture healing.In the fifth chapter, the aim of this part was to investigate the biomarkers in theserum of transient ischemic attack (TIA) patients by gel-based comparative proteomeanalysis. TIA is a strong predictor of subsequent stroke. In spite of having advancedimaging techniques, it is still challengeable to diagnose the early TIA in clinics. Weused gel-based comparative proteomic technique to screen the differentially expressedproteins in the serum of TIA patients.5differential proteins were successfullyidentified with the peptide mass fingerprint (PMF) method. Furthermore,2of5differential proteins were confirmed in serum from TIA patients and rats. Hence, webelieve that the exploration of these differentially expressed proteins or theircombinations with existent diagnostic tests may greatly improve the accuracy of earlydiagnosis of TIA and prevention during the short window between TIA and stroke.However, diagnostic tests are still needed to evaluate the sensitivity and specificity ofthese markers and also the number of false positives and false negatives.