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Effect of Platelet-activating Factor on Src-suppressed C Kinase Substrate Gene Expression in Rat Pulmonary Microvascular Endothelial Cells

Author: ChenShan
Tutor: SunGengYun
School: Anhui Medical University,
Course: Internal Medicine
Keywords: Platelet-activating factor Vascular endothelial cells Lung Inflammation Src inhibition of protein kinase C substrate Rats In situ hybridization
CLC: R655.3
Type: Master's thesis
Year: 2010
Downloads: 37
Quote: 0
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Abstract


Objective: To study the platelet-activating factor (platelet-activating factor, PAF) on cultured rat pulmonary microvascular endothelial cells (rat pulmonary microvascular of endothelial cells, RPMVEC) inhibition of Src protein kinase C substrate (Src-suppressed C kinase substrate with of SSeCKS ) mRNA expression observed signal pathway inhibitors SSeCKS mRNA expression of inflammation-mediated intervention role, combined with our previous work to further investigate the PAF to participate in the mechanism of acute lung injury (acute lung injury, ALI) and initially carried out SSeCKS in signal transduction pathways in gene expression in the inflammation induced RPMVEC injury research. Methods: MSCs isolated the culture RPMVEC; establish RPMVEC SSeCKS mRNA expression in situ hybridization detection methods; PAF stimulate the differences in time and concentration will RPMVEC randomly divided into the PAF dose group and the aging group: dose group were 10-10, 10-9,10-8,10-7 mol / L PAF, and RPMVEC role for 1.5 h the limitation group to 10-7 mol / L PAF and RPMVEC role 0.5,1.5,3,6,12,24 h, respectively; signaling pathway inhibitor intervention: RPMVEC 10μmol / L of nuclear transcription factor-κB (nuclear factor-kappa B, NF-κB) inhibitor pyrrolidine dimercapto ammonia acid (pyrrolidine dithiocarbamate, PDTC) pre-incubated for 1 h or 10μmol / L protein kinase C ( protein kinase C, PKC) inhibitor double indolyl maleimide (the bis-indolylmaleimide, BIM) pre-incubation of 0.5 h with 10-7 mol / L PAF or 10 mg / L lipopolysaccharide (lipopolysaccharide, LPS) for 1.5 h, the above are set properly, negative and positive control group; combined with in situ hybridization (in situ hybridization, ISH) with computer image analysis system software use to detect The different conditions RPMVEC SSeCKS mRNA expression changes. Results: 1. Vitro successful separation culture of RPMVEC, binding experiments confirmed by morphological and FITC-BSI. Successfully established digoxigenin-labeled oligonucleotide probes in situ hybridization detection RPMVEC SSeCKS mRNA in experimental methods. 3 normal RPMVEC have SSeCKS mRNA expression of a small amount, for brown finely granular hybridization signal was distributed in the cytoplasm. 10-10,10-9,10-8,10-7 mol / L PAF were incubated RPMVEC 1.5 h of SSeCKS mRNA expression levels with its concentration increases gradually increased, the differences were statistically significant compared with the control group. 10-7 mol / L PAF stimulate RPMVEC 0.5 h of SSeCKS mRNA expression levels that significantly increased, 1.5 h, reached the peak, then gradually decreased to 24 h, still higher than the normal control group. 4. PDTC significantly lowered PAF or LPS induced effects RPMVEC SSeCKS mRNA, while BIM this effect of non-intervention. Conclusion: 1. The successful RPMVEC vitro primary culture and identification. 2 by a combination of the use of the the ISH and computer image analysis system software successfully detected under different conditions RPMVEC SSeCKS gene expression changes. PAF was concentration and time dependent manner raised the level of the RPMVEC SSeCKS gene transcription. NF-κB signaling pathway rather than PKC involved in the PAF, LPS induced SSeCKS gene expression, signal transduction mechanisms.

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