Experimental And Clinical Research Of LPS Receptor TLR4,CD14 And Inflammatory Response

Part 1 : Research of the LPS receptor TLR4 "Asp299Gly" polymorphismObjective Genome research discover a missense mutation (Asp299Gly) in the fourthexon of TLR4, which results in replacement of a conserved aspartic acid residue withglycine at amino acid 299 and alters the extracellular domain of this receptor. Someresearch indicate that the Asp299Gly polymorphism may predispose people todevelop septic shock with gram-negative microorganisms. It is necessary to developea simple, accurate and practical method to detect the TLR4 Asp299Glypolymorphism , so we try to establish a new method based on allele specific PCR.Method Two allele specific primers ,with a mismatch introduced at the third3′-termminal base ,were both included in PCR system . The recombinant plasmidcarrying TLR4 polymorphism "Asp299Gly" or wild type TLR4 gene segmentconstructded through PCR site-directed mutagenesis were used as template to verifythe specificity of "Asp299Gly" Results 92 Chinese DNA sample were genotypedwith this method successfully Conclusion Our method of "ASPCR" was provedto be a rapid and simple technique for TLR4 polymorphism "Asp299Gly"genotyping and suitable for routine laboratories and large-scale population studies.But the "Asp299Gly" allele of the TLR-4 gene was not detected in any of the 92specimens.Part 2. Research of inflammatory response in LPS-stimulated whole blood cell in vitroObjective To establish a study model of human inflammatory response inLPS-stimulated whole blood cell in vitro. To observe the influence of variousconcentration of LPS on the expression of TLR4 and CD14 ,and the influence on theproduction of cytokine TNFα,IL-6,IL-10. Method Blood samples were drawninto tubes containing heparin and diluted with RPMI 1640, One milliliter of dilutedblood per well was placed into 24-well tissue culture plates. After different doses of -6-第二军医大学 英文摘要 硕士学位论文LPS added to each well, whole blood was stimulated with LPS (0–1000ng/mL).Measurements of cytokines were performed using ELISA. Expression of monocytecell surface TLR4 and CD14 was measured by flow cytometry. Results 1.Nocytokine was measured in the control group without LPS. 2. LPS induced TNFα,IL-6,IL-10 production in human whole blood in a dose-dependent manner atconcentrations of 0.01–10 ng/mL. TNFα,IL-6,IL-10 production reached a plateauwith LPS doses >10 ng/mL. 3. Expression of monocyte cell surface CD14 wasdownregulated by LPS at the concentration of 0.1ng/mL and 10 ng/mL. 4. Expressionof monocyte cell surface TLR4 was upregulated by LPS at the concentration of0.1ng/mL and 10 ng/mL. Conclution 1.Cytokine response can be actived with LPSdose > 0.01ng/mL in human whole blood in intro. TNFα,IL-6,IL-10 production inhuman whole blood in a dose-dependent manner at LPS concentrations of 0.01–10ng/mL.2. LPS can downregulate the expression of CD14 on monocyte cell surfaceand upregulate TLR4 at certain concentration.Part 3. Change of LPS receptor TLR4 , CD14 expression level and cytokine during the perioperative periodObjective Cytokine response during the perioperative period has been proved to becorrelated with postoperative recovery . LPS receptor TLR4,CD14 are the initial keymoleculer of human inflammatory response, so it is important to explore their changeduring the perioperative period and the regulation mechanism, and which may provideus new idea to deal with critical disease such as sepsis and MODS.Method Eight patients with liver tumors, four men and four women, were includedin the prospective study. The patients were admitted to the hospital for elective partialhepatectomy, classified as ASA physical status I and II, we obtained blood samplespre-anesthesia (T1), 45minute...