A multiple-antigen detection assay for tuberculosis diagnosis based on broadly reactive polyclonal antibodies.

OBJECTIVES: Detection of circulating Mycobacterium tuberculosis (M. tuberculosis) antigens is promising in Tuberculosis (TB) diagnosis. However, not a single antigen marker has been found to be widely expressed in all TB patients. This study is aimed to prepare broadly reactive polyclonal antibodies targeting multiple antigen markers (multi-target antibodies) and evaluate their efficacies in TB diagnosis. MATERIALS AND METHODS: A fusion gene consisting of 38kD, ESAT6, and CFP10 was constructed and overexpressed. The fusion polyprotein was used as an immunogen to elicit production of multi-target antibodies. Their reactivities were tested. Then, the multi-target antibodies and three corresponding antibodies elicited by each single antigen (mono-target antibodies) were evaluated with sandwich ELISA for detecting M. tuberculosis antigens. Their diagnostic efficacies for TB were also compared. RESULTS: The polyprotein successfully elicited production of multi-target antibodies targeting 38kD, ESAT6, and CFP10 as analyzed by Western blotting. When used as coating antibodies, the multi-target antibodies were more efficient in capturing the three antigens than the corresponding mono-target antibodies. By testing clinical serum, the multi-target antibodies demonstrated significantly higher sensitivity for clinical TB diagnosis than all three mono-target antibodies. CONCLUSION: The multi-target antibodies allowed detecting multiple antigens simultaneously and significantly enhanced TB detection compared to routine mono-target antibodies. Our study may provide a promising strategy for TB diagnosis.

Identification of Novel SCIRR69-Interacting Proteins During ER Stress Using SILAC-Immunoprecipitation Quantitative Proteomics Approach.

Spinal cord injury and regeneration-related protein #69 (SCIRR69),also known as cAMP-responsive element-binding protein 3-like 2, belongs to the CREB/ATF family, some members of which play significant roles in ER stress. However, it is still not fully elucidated whether SCIRR69 involves in ER stress and its biochemical and functional roles during ER stress. In this study, we firstly treated fetal rat spinal cord neuron cells (SCN) and PC12 cells with ER stress activator thapsigargin (TG) or tunicamycin (TM) and then detected the expression pattern of SCIRR69 in response to ER stress at mRNA and protein levels using real-time PCR assay and immunoblotting. Results showed that the expression pattern of SCIRR69 was largely consistent with those of ER stress marker (ATF6, BIP and CHOP) at either mRNA level or protein level, implying that SCIRR69 may play important roles in ER stress. Subsequently, we used stable isotope labeling by amino acids in cell culture (SILAC)-immunoprecipitation quantitative proteomics to identify interaction partners of SCIRR69 during TG-induced ER stress in PC12 cells and found that transitional endoplasmic reticulum ATPase (TERA) and sideroflexin-1 (SFXN1) were potential SCIRR69-interacting proteins. The interaction between SCIRR69 and TERA or SFXN1 was validated using co-immunoprecipitation. Those results provide some clues for novel signaling nexuses that made by interactions between SCIRR69 and TERA or SFXN1. Our findings may facilitate a better understanding of the fundamental functions of SCIRR69 during ER stress.

Identification of Novel RD1 Antigens and Their Combinations for Diagnosis of Sputum Smear-/Culture+ TB Patients.

Rapid and accurate diagnosis of pulmonary tuberculosis (PTB) is an unresolved problem worldwide, especially for sputum smear- (S-) cases. In this study, five antigen genes including Rv3871, Rv3874, Rv3875, Rv3876, and Rv3879 were cloned from Mycobacterium tuberculosis (Mtb) RD1 and overexpressed to generate antigen fragments. These antigens and their combinations were investigated for PTB serodiagnosis. 298 serum samples were collected from active PTB patients, including 117 sputum smear+ (S+) and sputum culture+ (C+) cases, 101 S-/C+ cases, and 80 S-/C- cases. The serum IgG levels of the five antigens were measured by ELISA. Based on IgG levels, the sensitivity/specificity of Rv3871, Rv3874, Rv3875, Rv3876, and Rv3879 for PTB detection was 81.21%/74.74%, 63.09%/94.78%, 32.21%/87.37%, 62.42%/85.26%, and 83.56%/83.16%, respectively. Furthermore, the optimal result for PTB diagnosis was achieved by combining antigens Rv3871, Rv3876, and Rv3879. In addition, the IgG levels of Rv3871, Rv3876, and Rv3879 were found to be higher in S-/C+ PTB patients than in other PTB populations. More importantly, combination of the three antigens demonstrated superior diagnostic performance for both S-/C+ and S-/C- PTB. In conclusion, the combination of Rv3871, Rv3876, and Rv3879 induced higher IgG response in sputum S-/C+ PTB patients and represents a promising biomarker combination for diagnosing of PTB.

Generation of monoclonal antibodies against MGA and comparison of their application in breast cancer detection by immunohistochemistry.

Mammaglobin A (MGA) is an organ specific molecular biomarker for metastatic breast cancer diagnosis. However, there are still needs to develop optimal monoclonal antibodies (mAbs) to detect MGA expression in breast carcinoma by immunohistochemistry. In this study, we first generated mAbs against MGA. Then, we used epitope prediction and computer-assisted structural analysis to screen five dominant epitopes and identified mAbs against five epitopes. Further immunohistochemical analysis on 42 breast carcinoma specimens showed that MHG1152 and MGD785 had intensive staining mainly in membrane, while CHH11617, CHH995 and MJF656 had more intensive staining within the cytoplasm. MGA scoring results showed that MJF656 had the highest rate (92.8%) of positive staining among five mAbs, including higher staining intensity when compared with that of MHG1152 (p < 0.01) and CHH995 (p < 0.05) and the highest the mean percentage of cells stained among mAbs. Furthermore, we analyzed the relationship of positive staining rate by mAbs with patient clinical characteristics. The results suggest that MJF656 was able to detect MGA expression, especially in early clinical stage, low grade and lymph node metastasis-negative breast carcinoma. In conclusion, our study generated five mAbs against MGA and identified the best candidate for detection of MGA expression in breast cancer tissues.

Transcription factor SCIRR69 involved in the activation of brain-derived neurotrophic factor gene promoter II in mechanically injured neurons.

The spinal cord injury and regeneration-related gene #69 (SCIRR69), which was identified in our screen for genes upregulated after spinal cord injury, encode a protein belonging to the cAMP response element-binding protein (CREB)/ATF family of transcription factors. Our previous study showed that SCIRR69 functions as a transcriptional activator. However, the target gene regulated by SCIRR69 and its roles in injured neurons remain unknown. In this study, we showed that SCIRR69 is widely distributed in the central nervous system. Full-length SCIRR69 is an endoplasmic reticulum-bound protein. Following mechanical injury to neurons, SCIRR69 was induced and proteolytically cleaved by site-1 and site-2 proteases, and the proteolytically cleaved SCIRR69 (p60-SCIRR69) was translocated to the nucleus where it bound to brain-derived neurotrophic factor (BDNF) gene promoter II. In addition, loss- and gain-of-function studies confirmed that SCIRR69 is involved in the regulation of BDNF expression in injured neurons. As expected, the culture supernatants of PC12 cells stably expressing p60-SCIRR69 contained higher levels of BDNF, and more remarkably promoted neurite outgrowth in a spinal cord slice culture model in vitro than the supernatants of control cells. These results suggest that SCIRR69 is a novel regulator of the BDNF gene and may play an important role in the repair and/or regeneration of damaged neural tissues by specifically activating BDNF promoter II.

Elevated serum haptoglobin after traumatic brain injury is synthesized mainly in liver.

Haptoglobin (Hp), an acute-phase response protein, is typically increased in the serum of adults after acute tissue injury. It is an antioxidant and may function as an injury-induced neuroprotective protein. However, the source of increased Hp is not clear. To investigate its source, we compared its time course expression profile in serum from rats with or without traumatic brain injury (TBI). Elevated Hp levels revealed by proteomic analysis were confirmed by Western blot, semiquantitative PCR, and real-time PCR. We found that Hp protein and mRNA levels were increased after TBI in both serum and liver, especially in liver. Both in vivo and in vitro data showed that Hp expression was increased in rat and human (HL7702) liver cells upon treatment with TBI serum. Addition of anti-interleukin-6 (IL-6) antibody downregulated the expression of Hp in liver cells induced by serum derived from rats and in liver of rats after TBI. These findings suggest that the increased Hp in serum came from the liver in response to TBI and that IL-6 is an important mediator of this induction.

Arachidonic acid: a bridge between traumatic brain injury and fracture healing.

Traumatic brain injury (TBI) is associated with enhanced osteogenesis. The aim of this study was to investigate the effect of serum from TBI rats on fracture healing. Results from this study showed that the serum from TBI rats enhanced the expression of bone gamma carboxyglutamate protein (BGLAP), and promoted in vitro proliferation of MC3T3-E1 cells, a mouse osteoblastic cell line. Furthermore, gas chromatography/mass spectrometry (GC/MS) coupled with multivariate statistical analysis was used to identify the changes in global serum metabolites after TBI. We found that arachidonic acid (AA) was significantly enhanced in serum metabolites in TBI subjects, while hydroxybutyric acid, leucine, malic acid, 5-oxyproline, isocitric acid, mannose, and uric acid were reduced. Finally, we examined the effects of AA on BGLAP expression and cell proliferation in MC3T3-E1 cells. We found that BGLAP expression and proliferation of osteoblasts were positively regulated in the presence of AA. These findings suggest that the increased AA in serum after TBI may play a key role in enhancing the speed of fracture healing.

Cloning and characterization of SCIRR69: a novel transcriptional factor belonging to the CREB/ATF family.

The complete cDNA sequence of a novel gene, SCIRR69 (spinal cord injury and regeneration related no. 69 gene), was obtained by RACE technique. It codes for a protein of 521 amino acid residues homologous to human CREB3l2 (also known as BBF2H7) and mouse CREB3l2. The protein contains a basic DNA binding and leucine zipper dimerization (B-ZIP) motif and a hydrophobic region representing a putative transmembrane domain, similar to the structure of other CREB/ATF transcription factors. Monoclonal antibody against SCIRR69 was developed and could recognize the SCIRR69 protein in both native and denatured forms. Constructing of SCIRR69 fusion proteins with the GAL4 DNA-binding domain disclosed that SCIRR69 functioned as a transcriptional activator and its N-terminal 60 amino acids accounted for the activation ability. SCIRR69 resides in the cytoplasm of primary neurons, whereas neuron damage by incision led to the cleavage and translocation from the cytoplasm to the nucleus. These results suggest that SCIRR69 is activated by proteolytic cleavage at the transmembrane domain in response to neuron damage and its amino-terminal cytoplasmic domain translocates into the nucleus to activate the transcription of target genes.

Relative expression of type II MAGE genes during retinoic acid-induced neural differentiation of mouse embryonic carcinoma P19 cells: a comparative real-time PCR analysis.

In mammals, the type II melanoma antigen (MAGE) protein family is constituted by at least ten closely related members, but our understanding of their function in the developing nervous system remains poor. To systematically study the expression pattern of type II MAGE genes during neurogenesis, we employed mouse embryonic carcinoma P19 cells as an in vitro model for neural differentiation by retinoic acid (RA) induction. The expression of type II MAGE genes was investigated under distinct steps of differentiation by a comparative ΔΔC (T) paradigm of real-time quantitative reverse-transcription PCR (qRT-PCR). The relative levels of each gene expression at various steps of differentiation were expressed as a fold change compared with that in RA-untreated P19 cells. The results revealed that: (1) the expression of MAGE-E1, E2, and Necdin transcripts was steadily increased, and the relative levels of MAGE-D1, D2, D3, F1, G1, and H1 mRNA were fluctuantly elevated after the RA-treatment at embryoid body and neural stages; (2) during RA-treatment and subsequent differentiation, the expression of MAGE-L2 mRNA was decreased. Therefore, our results suggested that MAGE-D1, D2, D3, E1, E2, F1, G1, H1, and Necdin might be involved in the early process of neurogenesis, and MAGE-L2 connected with maintenance of pluripotency of stem cells. These studies may present some clues for a better understanding of the fundamental aspects of type II MAGE genes during neurogenesis.

Establishment and assessment of a simple and easily reproducible incision model of spinal cord neuron cells in vitro.

A growing number of in vitro models have been introduced to study the mechanisms of spinal cord injury. A potential drawback of these models is that they are difficult to reproduce. In this study, an in vitro incision model was established using primary cultured neuronal cells from fetal rat spinal cords. The neurons were subjected to incision in a simple and reproducible way. To assess whether this model could simulate the responses of spinal cord neuron cells in vivo after a spinal cord transection, apoptosis, and the expression of immediate early genes were detected in the neurons at various time points after injury. The results indicated that: (1) significantly more terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells were observed at 1, 3, and 7 d following injury and (2) the expression of both c-Jun and c-Fos was induced 10 min after incision and had markedly higher levels 2 h post-injury. These results suggested that our model can partially imitate the responses of in vivo neuronal cells after a spinal cord transection and such models may facilitate further understanding of biochemical and cellular events associated with spinal cord injury.

Preparation and characterization of murine monoclonal antibodies against rat spinal cord injury and regeneration related protein no. 69.

Spinal cord injury and regeneration related protein No. 69 (SCIRR69) is a rat transmembrane bZIP transcription factor homologous to mice and human transcription factor CREB3L2. Previous work demonstrated the N-terminal region plays a critical role in its transcriptional activity. In this study, a peptide containing 18 amino acids (5-22aa) at the N-terminus of rat SCIRR69 was synthesized and coupled to the carrier protein as immunogen. One hybridoma cell line was obtained by standard cell fusion technique, followed by enzyme-linked immunosorbent assay (ELISA) and Western blot screening. The newly developed monoclonal antibody (MAb) was designated 4B4, the isotype of which was IgG2a. Immunofluorescence and Western blotting results showed that MAb 4B4 could recognize the SCIRR69 protein in both native and denatured forms. 4B4 will be a useful tool for the functional research of SCIRR69 in future studies.

Proteomic profiling of the insoluble pellets of the transected rat spinal cord.

This study investigated the proteomic changes at different time points in the precipitated pellets of rat spinal cords after applying complete spinal cord transection. By two-dimensional electrophoresis, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, MALDI-TOF/TOF and peptide mass fingerprinting analysis, 44 proteins were identified, most of which are membrane and/or organellar proteins. They are mainly involved in metabolic processes (75%), developmental processes (30%), or responses to stimuli (30%), playing negative or positive roles. In particular, decreases of pyruvate dehydrogenase beta, aconitase 2, fumarate hydratase 1, and ATP synthase subunit 6 can lead to ATP depletion by crippling tricarboxylic acid cycle and oxidative phosphorylation. Decreases of several antioxidant proteins such as catalase, peroxiredoxin 1, Parkinson disease 7, and stress-induced phosphoprotein 1 can contribute to the secondary injury of spinal cord. Decreases of development-related 3-phosphoglycerate dehydrogenase and stathmin 1 may be not propitious for spinal cord regeneration. On the other hand, increases of isocitrate dehydrogenase 3 alpha/gamma and glutamate dehydrogenase 1 can help compensate the impaired energy metabolism. Increases of sirtuin 2, crystallin alpha B (CRYAB), and heat shock 27-kDa protein 1 can help resist stresses induced by injury. Increases of adenylate cyclase-associated protein 1 and galactose binding lectin 3 can help regeneration by replaying their roles in neural development. To our knowledge, this is the first case of characterization of the proteomic changes seen in the precipitated fraction of injured spinal cord. Most of the identified proteins were found for the first time to be differentially expressed after spinal cord injury, which may provide new clues about the molecular mechanisms of spinal cord injury and repair.

GPI-1046 stimulates chicken dorsal root ganglion neurite outgrowth in the presence of nerve growth factor at low concentration in vitro.

The purpose of this investigation was to re-evaluate the neurotrophic effect of GPI-1046 on neurite outgrowth in vitro. GPI-1046 was synthesized and identified with mass spectrometry, nuclear magnetic resonance and elemental analysis. Chicken dorsal root ganglions (DRGs) were removed and divided into three groups: (1) The DRGs were cultured in DMEM containing different concentrations of GPI-1046; (2) The DRGs were cultured in DMEM containing nerve growth factor (NGF) alone at 0.8 and 8 ng/mL, respectively; (3) The DRGs were cultured in DMEM containing both different concentrations of GPI-1046 and NGF at 0.8 ng/mL. The results showed that GPI-1046 alone could not stimulate chicken DRG neurite outgrowth; however, GPI-1046 stimulated DRG neurite outgrowth only in the presence of NGF at low concentration in the culture medium.

Identification and characterization of scirr1, a novel gene up-regulated after spinal cord injury.

Spinal cord injury and regeneration involves transcriptional activity of many genes, of which many remain unknown. Using the rat spinal cord full- transection model, bioinformatics, cloning, expression assays, fusion proteins, and transfection techniques, we identified and characterized one such differentially expressed gene, termed scirr1 (spinal cord injury and/or regeneration related gene 1). Fourteen orthologs were found in 13 species from echinoderm to insect and human by Blast search of NCBI protein reference sequence database. However, no further information is available for these homologues. Using whole-mount in situ hybridization, mouse scirr1 mRNA was expressed temporally and spatially in accordance with the early development sequence of the central nervous system. In adult rat spinal cord, expression of scirr1 mRNA was localized to neurons of gray matter by in situ hybridization. Using immunohistochemistry, SCIRR1 protein was found to be up-regulated and expressed more highly in spinal cord neurons farther from the epicenter of injury. Although the precise function of SCIRR1 is unknown, its unique pattern of expression during CNS early development and up-regulation after spinal cord injury suggest that SCIRR1 should be involved in the succeeding injury and/or repair processes of the injured spinal cord. Also, the typical F-box and leucine-rich repeat (LRR) architecture of rat SCIRR1 indicated that it may play an important substrate recruiting role in the pleiotropic ubiquitin/proteasome pathway. All these make scirr1 a new interesting start to study the spinal cord injury and regeneration mechanism.

Comparative proteomic study of acute pulmonary embolism in a rat model.

Pulmonary embolism (PE) is a common, potentially fatal disease, whose blood clots originate from the deep venous system of the lower extremities. PE is of clinical importance because of the considerable mortality and morbidity. In this study, at first we established a rat PE model by injecting 3-4 emboli into the left jugular vein. Before collecting the lung tissues, we perfused them with saline through the right jugular vein and at the same time cut off the right carotid to remove the blood. Then we separated and identified differentially expressed proteins in lung tissues at different time points using the techniques of 2-DE and MS. After image analysis of 2-DE gels, 46 protein spots of interest were excised from the gels and identified by MALDI-TOF-MS. Thirty-two protein spots of them found their corresponding protein candidates in the database. These proteins are associated with distinct aspects of PE such as the contractive function of smooth muscles, metabolism of energy, collagen and toxicant, cellular differentiation, apoptosis and injury, blood pressure adjustment, maintaining of acid-base balance, and so on. Ten of the identified proteins were validated by semiquantitative RT-PCR, and three of them were further validated by Western blot analysis. The differential expression patterns of these proteins suggest the distinct roles they may play in different stages of the rat PE model, and information from this study may be helpful to uncover the pathophysiologic molecular mechanisms involved in PE.

Comparative proteome analysis of serum from acute pulmonary embolism rat model for biomarker discovery.

Pulmonary embolism (PE) is a common, potentially fatal disease and its diagnosis is challenging because clinical signs and symptoms are nonspecific. In this study, to investigate protein alterations of a rat PE model, total serum proteins collected at different time points were separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Bioinformatics analysis of 24 differentially expressed proteins showed that 20 had corresponding protein candidates in the database. According to their properties and obvious alterations after PE, changes of serum concentrations of Hp, Fn, DBP, RBP, and TTR were selected to be reidentified by western blot analysis. Semiquantitative RT-PCR showed DBP, RBP, and TTR to be down-regulated at mRNA levels in livers but not in lung tissues. The low serum concentrations of DBP, RBP, and TTR resulted in the up-regulation of 25(OH)D3, vitamin A, and FT4 (ligands of DBP, RBP, and TTR) after acute PE in rat models. The serum levels of Hp and Fn were detected in patients with DVT/PE and controls to explore their diagnostic prospects in acute PE because the mRNA levels of Hp and Fn were found to be up-regulated both in lung tissues and in livers after acute PE. Our data suggested that the concentration of serum Fn in controls was 79.42 +/- 31.57 microg/L, whereas that of PE/DVT patients was 554.43 +/- 136.18 microg/L (P < 0.001), and that the concentration of serum Hp in controls was 824.37 +/- 235.24 mg/L, whereas that of PE/DVT patients was 2063.48 +/- 425.38 mg/L (P < 0.001). The experimental PE rat model selected in this study was more similar to the clinical process than the other existing PE animal models, and the findings indicated instant changes of serum proteins within 48 h after acute PE. The exploration of these differentially expressed proteins or their combination with existent markers such as D-dimer may greatly improve the accuracy of the diagnosis of acute PE, but diagnostic tests are still needed to evaluate the sensitivity and specificity of these markers and also the number of false positives and false negatives.

Neurons derived from PC12 cells have the potential to develop synapses with primary neurons from rat cortex.

Neuron transplantation is considered to be a promising therapeutic method to replace functions lost due to central nervous system (CNS) damage. However, little is known about the extent to which implanted neuron-like cells can develop into mature neurons and acquire essential properties, and especially formation of synapses with host neurons. In this investigation we seeded PC12 cells labeled with GFP into primary cultured neurons isolated from rat cerebral cortex to build a co-culture system, and then induced the PC12 cells to differentiate into neuron-like cells with NGF. Seven days later, we observed the relationship between the PC12-derived neurons and primary neurons using FM1-43 imaging and immunoelectron microscopy, and found that GFP-labeled neurons could form typical synapses with host primary neurons. These observations showed that immigrant neurons differentiated from PC12 cells could develop into mature neurons and could form intercellular contacts with host neurons. Both the immigrant and host neurons could construct neuronal networks in vitro.

Identification of up-regulated genes after complete spinal cord transection in adult rats.

Spinal cord injury (SCI) initiates a cascade of events and these responses to injury are likely to be mediated and reflected by changes in mRNA concentrations. As a step towards understanding the complex mechanisms underlying repair and regeneration after SCI, the gene expression pattern was examined 4.5 days after complete transection at T8-9 level of rat spinal cord. Improved subtractive hybridization was used to establish a subtracted cDNA library using cDNAs from normal rat spinal cord as driver and cDNAs from injured spinal cord as tester. By expressed sequence tag (EST) sequencing, we obtained 73 EST fragments from this library, representing 40 differentially expressed genes. Among them, 32 were known genes and 8 were novel genes. Functions of all annotated genes were scattered in almost every important field of cell life such as DNA repair, detoxification, mRNA quality control, cell cycle control, and signaling, which reflected the complexity of SCI and regeneration. Then we verified subtraction results with semiquantitative RT-PCR for eight genes. These analyses confirmed, to a large extent, that the subtraction results accurately reflected the molecular changes occurring at 4.5 days post-SCI. The current study identified a number of genes that may shed new light on SCI-related inflammation, neuroprotection, neurite-outgrowth, synaptogenesis, and astrogliosis. In conclusion, the identification of molecular changes using improved subtractive hybridization may lead to a better understanding of molecular mechanisms responsible for repair and regeneration after SCI.

Proteome analysis of up-regulated proteins in the rat spinal cord induced by transection injury.

The inability of the CNS to regenerate in adult mammals propels us to reveal associated proteins involved in the injured CNS. In this paper, either thoracic laminectomy (as sham control) or thoracic spinal cord transection was performed on male adult rats. Five days after surgery, the whole spinal cord tissue was dissected and fractionated into water-soluble (dissolved in Tris buffer) and water-insoluble (dissolved in a solution containing chaotropes and surfactants) portions for 2-DE. Protein identification was performed by MS and further confirmed by Western blot. As a result, over 30 protein spots in the injured spinal cord were shown to be up-regulated no less than 1.5-fold. These identified proteins possibly play various roles during the injury and repair process and may be functionally categorized as several different groups, such as stress-responsive and metabolic changes, lipid and protein degeneration, neural survival and regeneration. In particular, over-expression of 11-zinc finger protein and glypican may be responsible for the inhibition of axonal growth and regeneration. Moreover, three unknown proteins with novel sequences were found to be up-regulated by spinal cord injury. Further characterization of these molecules may help us come closer to understanding the mechanisms that underlie the inability of the adult CNS to regenerate.

cDNA microarray analysis of spinal cord injury and regeneration related genes in rat.

The acute traumatic spinal cord injury (SCI) is a commonly seen and severe case in clinic. However, the repair and regeneration of injured spinal cord is limited. This is likely due to that different kinds of factors are involved in regeneration after SCI. In the present study, we used complementary DNA microarray consisting of 4 041 specific probes from rat to identify genes that were differentially expressed after SCI. The animals were subjected to complete transection injury of the thoracic spinal cord (T8-T9). Sham operated animals received only a laminectomy. Four and a half days later, rat spinal cord was dissected out for total RNA isolation. The fluorescent (Cy3 and Cy5) labeled probes were prepared and hybridized to the microarray. Genes that showed 2-fold difference in SCI tissue were identified. Sixty-five up-regulated genes consisted of 21 known genes, 30 known expressed sequence tags (ESTs) and 14 unknown genes. Seventy-nine down-regulated genes comprised 20 known genes, 42 known ESTs and 17 unknown genes. In 41 differentially expressed known genes, 5 up-regulated genes, i.e., tissue inhibitor of metalloproteinase 1 (Timp1), transgelin (Tagln), vimentin (Vim), Fc gamma receptor, cathepsin S (Ctss), and 3 down-regulated genes, i.e., stearyl-CoA desaturase, coagulation factor II (F2), endosulfin alpha (Ensa), were further confirmed by reverse transcription polymerase chain reaction (RT-PCR). These genes may play a role in the response to tissue damage or repair following SCI and characterization of them might be helpful to elucidate the molecular mechanisms of spinal cord injury and regeneration.