| Severe Severe burns patients are susceptible to inflection, which often induces sepsis and septic shock. Septic shock is characterized by the appearance of hypotension that is resistant to volume replacement alone, and by organ hypoperfusion. The basic paradigm of septic shock is that microbial antigens such as Gram-negative bacterial Lipopolysaccharide (LPS), or endotoxin, initiate an uncontrolled network of host-derived proinflammatory mediators, which ultimately lead to cardiovascular shock and death.Endothelial cells (EC) play an important regulatory role in sepsis by upregulating various proinflammatory gene product, and the vascular change in septic shock is on the basis of the effect of inflammatory mediators on the vascular EC. It has been proposed that widespread EC activation or damage occurs during human sepsis and leads to organ dysfunction and failure.LPS, the major component of the outer surface of Gram-negative bacteria, is a potent activator of cells of the immune and inflammatory systems and contributes to the systemic changes seen in septic shock. LPS directly or indirectly evokes numerous EC responses. However, the metabolism of LPS on the direct activation is still unclear.Phenotype differences between cells of the same organism are determined by differential expression of identical genes. Identification of differentially expressed genes has been a valuable tool for understanding gene function and for investigating molecular mechanisms of LPS activates vascular EC. To investigate the influence of LPS on gene transcription in EC, In the studiesdescribed here, two-directional (forward and backward) suppression subtractive hybridization (SSH) was performed on human umbilical vein endothelial cells (HUVEC) cultured in either standard media or treated for 6 hours with LPS( 100ng/ml). With the T/A cloning method the cDNA fragments generated by SSH were cloned into pT-Adv plasmid, the subtracted cDNA libraries being constructed. To restrict the number of false-positive clones, colony array dot hybridization was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced and analysed in GenBank with Blast 2.0 search. The results showed that two high quality forward and backward subtracted cDNA libraries were constructed. Thirtye-one up-regulated expressed cDNA fragments and thirteen down-regulated cDNA fragments in HUVEC stimulated with LPS were identified, including forty known genes and four novel cDNA sequences ( ST40, ST55, ST67 and ST78 ). The known genes include a group related to proinflammatory events, a group of related to prothrombotic function, a group of related to cellular apoptosis, a group related to cytoskeletal rearrangment, a group related to energy metabolism, a group related to signal transduction and a group of unknown function proteins. Some of the identified cDNAs were corresponding to known LPS responsive genes such as E-selectin, but also the study revealed a number of mRNAs previously not known to be responsive to LPS such as laminin receptor and caveolin. The four novel cDNA sequences have been accessed as novel express sequence tags (EST) by GenBank (accessment No. BI850093, BM121646, BI850094, BI850055).The novel ESTs were analysed by bioinformatics. ST40 is highly homology with TAF 105 subunit, and ST67 is homology with focal adhesion kinase. ST78 and ST55 are not homology with any known gene of human, so they were amplificated by rapidly amplification of cDNA ends (RACE) to obtain the full-length cDNA sequences. A 1404bp novel cDNA with a poly(A)+IVtail and an open reading frame encoding a 158 amino acid protein was cloned from ST55. Northern blot indicated the novel cDNA sequence was rare expressed in unstimulated HUVEC but significantly up-regulated in HUVEC stimulated with LPS. The gene and the encoded protein were termed endothelial cell overexpressed LPS associated factorl (EOLAl) and EOLA1. In human multiple tissue Northern blot analysis, EOLAl transcription expresses in heart, skeletal muscle, kidney, l...
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