Study On Different Expressed Gene Between Rat Monocyte And Alveolar Macrophage Following Trauma With LPS Challenge

MODS (multiple organs dysfunction syndrome) is defined as altered organ function in the setting of sepsis, septic shock, or systemic inflammatory response syndrome. It is characterized by acute onset, rapid progress, and high mortality. It is related to systemic excessive inflammation and imbalance of oxygen intake and transmission ratio. The lung is an organ that filters systemic venous blood and metabolites. Pulmonary dysfunction is often a frequent complication following MODS. So acute respiratory distress syndrome (ARDS) is the often critical clinical situation following serious injury. Macrophages are the key cells that mediate innate and adaptive immunity; they are associated with an excessive inflammation response following severe sepsis. Normally, peripheral blood monocytes transform into resident tissue-specific macrophages through migration from blood vessels. During inflammation, macrophage migration and proliferation increases, resulting in relatively larger aggregations of these cells. Alveolar macrophages (AMs) are the first defense of the lung. They maintain immune homeostasis. Their traditional role is as a "professional" phagocyte, however, these cells can also produce numerous mediators including cytokines, growth factors, reactive oxygen species and arachidonic acid derivatives. AMs possess a unique phenotype compared with macrophages in other tissue compartments.It has been proven that peripheral blood monocytes and AMs are heterogeneous, they are correlated in differentiation and development, while they have different functions. Their heterogeneity plays an important role in immune dysfunction after trauma, while little is known about the changes in mRNA expression after trauma. The differences between peripheral blood monocytes and alveolar macrophages suggest that a comparison of mRNA expression between them would be helpful to understand their roles in immune dysfunction after serious trauma.In view of this, we established a trauma animal model by inflicting blunt injuries onwistar rat hind limbs and simultaneous LPS challenge. Switching Mechanism At 5' end of the RNA Transcript (SMART) and suppression subtractive hybridization (SSH) methods were used to construct differential expression cDNA libraries of peripheral blood monocyte and alveolar macrophage following trauma with LPS challenge. All positive clones have been sequenced, followed by BLASTN data analysis on Genbank. The significances of the differential mRNA expression between monocytes and alveolar macrophages from this injury animal model have been discussed.The main results and conclusions are as below:1. Combining SMART and SSH methods, we constructed differential expression cDNA libraries of peripheral blood monocytes and alveolar macrophages collected from the injured wistar rat 1 day after trauma. Eighty-nine clones of monocyte and 152 clones of alveolar macrophage are acquired.2. The specific PCR, PCR-Select Differential Screening, Northern Direct HRP labeling and other corresponding detection methods were used to screen and evaluate the differential expression cDNA libraries by forward-subtracted and reverse-subtracted methods. Fifty-eight positive clones were acquired in our study. Among them 15 sequences are from monocytes, and 43 sequences are from alveolar macrophages.3. After sequence and homologous analysis by NCBI BLASTN database, the results indicated that the sequences were highly homologous to genes known to be involved in diverse biological processes, including chemotaxis, energy metabolism, cell proliferation and differentiation, even signal transduction etc. This suggests that immune dysfunction after trauma is related to multiple signal transduction systems and a wide array of biochemical events.4. Two novel ESTs we submitted to GenBank were enrolled.