The Relation Between LPS And Expression Of TRAM Gene,and The Inhibiting Effect Of TRAM SiRNA On Inflammation Of LPS

Septic shock, one of the most severe complication, is deadly,its mortality varies from 35%-70%. Bacterial endotoxin or lipopolysaccharide (LPS) is one of the potent mediators in inducing sepsis or uncontrolled inflammatory response. Toll-like receptor4 (TLR4) is mostly the receptor for LPS, and serves as an important link between the innate and adaptive immune responses. Normally, the immune response tightly controls the local inflammatory process to eradicate invading gram-negative bacteria .When this control mechanism fails, systemic inflammatory response occurs, converting the infection to sepsis, or septic shock. Some mechanisms have continuously been exploring although the molecular mechanisms of inflammatory response induced LPS had been lucubrated. There are a great many targets on controlling inflammatory response of LPS in view of the molecular mechanisms of inflammation. It is very difficult to choose a target that can inhibit inflammation of LPS to the uncontrolled inflammatory response, little influences on person, and has the likelihood of clinic application. Recently, the MyD88-independent pathway of TLR4 signaling has been identified, and some research findings have showed that the MyD88-indepent pathway was of consequence to occurrence and deterioration on septic shock of endotoxin. The pathway might help discover some novel strategies for effective control or modulation of uncontrolled inflammatory response in the LPS response. It theoretically may meet the conditions of candidacy, as Toll/IL-1 receptor domain-containing adaptor inducing interferon-β(TRAM) is specifically involved in TLR4-mediated MyD88-independent signaling pathway. Therefore, this study chooses TRAM as the object to investigate the relation between LPS and expression of TRAM gene, and to investigate the inhibiting effect of TRAM siRNA on inflammation of LPS. The main methods and conclusions are as follows: 1. Based on amino acid sequence of TRAM protein, the analysis of the flexible regions of secondary structure and the B cell epitopes of TRAM protein were predicted by software of nnpredict and Goldkey. The computer predicted that most possible epitopes of TRAM protein were within or nearby its N-teminal No. 216-229, which accorded with its hydrophilicity, accessibility and flexibility, and which located in extension and/or rulelessly convolute frame of the protein for the secondary structure. The amino acid sequence is QSIWKETRSVSQKQ. 2. The peptide of the epitopes was synthesized and used for immunizing rabbits. The immunogenicity of the peptide was detected with ELISA. The specificity of polyclonal anti-TRAM was measured with immunocytochemistry assay and Western blotting. These findings indicate that synthesized peptides induced high titer of antibody and the polyclonal antibody to TRAM has high specificity. 3. The specific primer to TRAM gene was designed by general principle of polymerase chain reaction (PCR), and was expanded into the specific cDNA piece of TRAM gene. Then, the specific cDNA piece was inserted into pGEM?-T vector, and the recombinant pGME?-T vector was transformed into E.coli DH5a. The recombinant has been ratified by cutting of enzyme and sequencing of DNA. Therefore, the recombinant vector establishes the outer standard for real -time quantitative PCR (RQ-PCR) to determine mRNA expression of TRAM gene. 4. The expressed mRNA of TRAM gene was detected by RQ-PCR. There was the low expression of TRAM mRNA (about 2.51×105 copies number) in RAW264.7 cells. When RAW264.7 cells were continually stimulated with LPS (100ng/ml), the number of expression increased markedly at 4h (P<0.05), and sharply went up from 8h to 12h (P<0.01), peaked at approximately 3.46×106 copies number at 12h. Then, the number of expression steeply decrease from 12h to 24h, but was higher than control group at 24h. The expressed protein of TRAM gene was detected with immunocytochemistry assay and Western blotting. The statistics show that there are a lot of similarities between RQ-PCR and immunocytochemistry assay/Western blotting. The expression of protein reached peak at 16h in immunocytochemistry assay, at 12h in Western blotting. In addition, when RAW264.7 cells were stimulated with LPS 100ng/ml and 1000 ng/ml at 16h, the expression of protein in LPS 1000 ng/ml was greater than in 100 ng/ml. The data lead us to conclude that there are tight relation between LPS and expression of TRAM gene.5. The specific target sequence (Each oligo 64 nt) to mouse TRAM mRNA was designed by general principle of siRNA. To construct and identify a vector expressing TRAM siRNA in mammalian cells, R-pSUPER-EGFP vector used to transcribe functional TRAM siRNA were constructed. Two of pair 64 nt TRAM gene specific target sequences were inserted into the downstream of the H1 promoter. Two different recombinant plasmids containing corresponding TRAM gene specific target sequences were constructed. The recombinants were transformed into E. coli JM109, and finally confirmed their veracity by double cutting with the enzymes and sequencing of DNA. 6. R-pSUPER-EGFPs were transfected into RAW264.7 cell by using LipofectamineTM2000, and the expression of TRAM was detected by RQ-PCR and Western blotting. R-pSUPER-EGFPs were transfected into RAW264.7 cell line successively, and can specifically restrain expression of TRAM protein. Therefore, the recombinant vectors established the basis for research on the function of TRAM gene. 7. After wild RAW264.7 cells (WC) and RAW264.7 cell transfected R-pSUPER-EGFP (R1C and R2C) were continually stimulated with LPS (100ng/ml) at 2h, the activation of nuclear factor-kappa B (NF-кB) was detected with electrophoretic mobility shift assay (EMSA). The experiment indicated that R1C and R2C could significantly inhibit the activation of NF-кB. In the same way, ELISA indicated that R1C and R2C could significantly inhibit the excretion of TNF-α, IL-1Ra and INF-βfor LPS (100ng/ml) stimulation at 4h, 8h and 12h. These findings show that TRAM siRNA has the inhibiting effect on inflammation of LPS. Judging from all evidence offered, we may reasonably draw the following conclusions: (1) siRNA offers a useful laboratory tool in mammal cells; (2) TRAM siRNA has the inhibiting effect on inflammation of LPS in the level of cell; (3) TRAM gene could be a target to control or modulation of uncontrolled inflammatory response in the LPS response. It will require more alive body research to define whether siRNA TRAM has the likelihood of clinic application.