OBJECTIVETo investigate the silencing effect of gene encoding peroxisome proliferator-activated receptor gamma (PPARgamma) on the expression of tumor necrosis factor alpha (TNFalpha) by constructing vectors for RNA interference in RAW264.7 cells.

METHODSThe pSUPER-EGFP vectors were used to transcribe functional small interfering RNA (siRNA). Four pairs of oligonucleotides (64 nt) targeting PPARgamma gene were inserted into the downstream of the H1 promotor, with their veracity confirmed by double digestion and sequencing. Western blotting and immunofluorescence assay were used to examine the silencing effect of PPARgamma gene in RAW264.7 cells. Meanwhile, the TNFalphalevel was determined by Sandwich ELISA.

RESULTSCompared with other recombinant pSUPER-EGFP vectors (R-pSUPER.EGFP), R-pSUPER.EGFP2 induced the best silencing effect on the expression of PPARgamma in RAW264.7 cells, which played an obvious inhibitory role in down-regulating the TNFalphaexpression after the curcumin and lipopolysaccharide (LPS) stimulation.

CONCLUSIONSPPARgamma-pSUPER-EGFP inducing a silencing effect on the expression of PPARgamma can efficiently play a negative role in controlling the inflammatory responses of RAW264.7 cells.

Acute-Phase Proteins ; physiology ; Carrier Proteins ; physiology ; Humans ; Membrane Glycoproteins ; physiology ; Receptors, Cell Surface ; physiology ; Receptors, Immunologic ; physiology ; Receptors, Scavenger ; Sepsis ; metabolism ; physiopathology ; Signal Transduction ; Toll-Like Receptors

OBJECTIVETo investigate the frequencies of -1470, -511 and -31 single nucleotide polymorphisms (SNPs) in the promoter of IL-1beta and its haplotype constitution in Chongqing population.

METHODSOne hundred and twelve healthy Chongqing people were enrolled in this study. Polymorphisms at -1470 (G to C), -511 (T to C) and -31 (C to T) of IL-1beta were genotyped with the method of restriction fragment length polymorphism (RFLP). Haplotype frequencies were analyzed by Arlequine software.

RESULTSFrequencies of IL-1beta -1470, -511 and -31 SNPs were 41.67%, 50% and 45.33%, respectively. Genotype frequencies of -1470 locus were 39.81%, 37.04% and 23.15% for G/G, G/C and C/C respectively. As for T-511C SNP, genotype frequencies of T/T, T/C and C/C were 29.91%, 40.18% and 29.91%, respectively. Genotyping results of C/C, C/T, and T/T of -31 locus were 35.51%, 38.32% and 26.71% respectively. Haplotype analysis found that there were mainly three haplotypes constituted by three SNPs, ie., G-T-C, C-T-C and G-C-T.

CONCLUSIONSPolymorphisms exist in the promoter of IL-1beta in Chongqing population. Three SNPs locate in the same haplotype block.

Asian Continental Ancestry Group ; genetics ; China ; Cohort Studies ; Female ; Genetics, Population ; Genotype ; Haplotypes ; Humans ; Interleukin-1 ; genetics ; Male ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single-Stranded Conformational ; Promoter Regions, Genetic ; Sensitivity and Specificity

OBJECTIVETo investigate the frequencies of -31, -511 and -1470 single nucleotide polymorphisms (SNPs) in the promoter of IL-1 beta in Chongqing population and address the question whether they are in linkage disequilibrium (LD).

METHODSOne hundred and twelve healthy Chongqing people were enrolled in this study. Polymorphisms at -31 (C>T), -511 (T>C) and -1470 (G>C) of IL-1 beta were genotyped with the method of restriction fragment length polymorphism (RFLP). Haplotype frequencies were evaluated with Arlequine software. Two-point LD analyses were done with the software TransposerV1-0. The P values were determined by Pearson chi square test analysis.

RESULTSAllelic frequencies of IL-1 beta -31, -511 and -1470 were 45.33%, 50.00% and 41.67%, respectively. The dominant haplotypes comprising the three loci were T-C-G (44.1%), C-T-C(40.3%) and C-T-G(8.8%), LD analyses revealed that none of the LD parameters(delta value) was 0. Meanwhile, chi square test showed P<0.005.

CONCLUSION-31, -511 and -1470 loci in the promoter region of IL-1 beta are in strong linkage disequilibrium. And this study provides a basis for searching disease-related IL-1 beta haplotye.

Adult ; China ; Female ; Gene Frequency ; Genotype ; Haplotypes ; Humans ; Interleukin-1beta ; genetics ; Linkage Disequilibrium ; Male ; Middle Aged ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; genetics

OBJECTIVEEarly gestational mammalian fetuses possess the amazing ability to heal cutaneous wounds in a scarless fashion. Over the past years, scientists have been working to decipher the mechanisms underlying this regenerative repair. The remarkable phenotypic differences between fetal and adult healings behooves us to learn their characteristics in genetics, which represents potentially important mechanisms involved in wound repair observed in fetal versus adult tissues. In this sense, it is reasonable to construct subtractive cDNA library for future research.

METHODSMiddle laparotomy and hysterotomy were performed on pregnant rabbits at 20-day gestation to expose the fetal back, and a longitudinal incision through the skin was made on the back of the fetus. The traumatized fetal skin was harvested 12 hours post-operation, the fetus control and traumatized adult skin specimens were taken at the same time. dscDNA was synthesized from total RNA of skin samples with SMART technology. Taking one of the three samples as Tester respectively and the other two as Drivers, we obtained 1 forward and 2 reverse hybridization products. After being amplified with selective polymerase chain reaction, the products were inserted into a vector, and then transferred into E. coli HB101. The colonies were screened afterwards.

RESULTSThe wounded fetuses were alive for a long time even after birth. Every determinant step, such as RNA isolation, cDNA synthesis, Rsa I digestion, adaptor ligation and hybridization, was well-operated. Subtractive efficiency identification demonstrated that the suppression subtractive hybridization (SSH) was successful. Insertion into vector and transferring to E. coli were satisfactory.

CONCLUSIONSInstead of classic SSH, an improved SSH with 2 Drivers was applied for the experiment. Results confirmed that the improved program was reasonable and correct in both theory and practice. The subtractive cDNA library we have obtained is going to be used for future researches to reveal scarless healing related gene(s) and its (their) expression.

Animals ; Cicatrix ; genetics ; DNA, Complementary ; Disease Models, Animal ; Female ; Fetus ; Gene Library ; Polymerase Chain Reaction ; Pregnancy ; Pregnancy, Animal ; RNA, Messenger ; analysis ; Rabbits ; Sensitivity and Specificity ; Skin ; injuries ; Wound Healing ; genetics ; physiology

OBJECTIVETo investigate the expression of myeloid differentiation protein-2 (MD-2) in the human endothelial cells and its role in lipopolysaccharide (LPS)-induced NF-kappaB activation in endothelial cells.

METHODSIn vitro cultured human umbilical vein endothelial cells (HUVEC) were employed in the study. The expression of MD-2 mRNA and protein, and the effect of LPS on the expression of its mRNA and protein were assessed with RT-PCR and Western blotting. The role of MD-2 in LPS-induced NF-kappaB activation and IL-8 production were investigated with gene transfection of mutant MD-2 cDNA (0.5, 1.0, 2.0 microg), pEF-BOS vacant vector (2.0 microg) and MD-2 plasmid (2.0 microg) into HUVEC, respectively.

RESULTSThere was MD-2 mRNA and protein expression in HUVECs before LPS stimulation, and it could be obviously upregulated by LPS in time and dose-dependent manner (MD-2 protein absorbency was 25 196 +/- 1 723 without LPS stimulation, which was obviously lower than that stimulated with 0.01 mg/L LPS (58 817 +/- 3 241, P < 0.01) for 6 hours. Transfection of mutant MD-2 cDNA could remarkably inhibit LPS-induced NF-kappaB activation and IL-8 production in endothelial cells.

CONCLUSIONMD-2 might play an important role in the LPS-induced NF-kappaB activation in HUVECs.

Cell Differentiation ; Cells, Cultured ; Endothelial Cells ; metabolism ; Humans ; Interleukin-6 ; genetics ; Interleukin-8 ; metabolism ; Lipopolysaccharides ; pharmacology ; NF-kappa B ; metabolism ; Umbilical Veins ; cytology

OBJECTIVETo construct the multi-probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD-2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells.

METHODSThe designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti-sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method.

RESULTSThe proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD-2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1-3 hours after 100 ng/ml LPS stimulation.

CONCLUSIONSThese new RPA multi-probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD-2 mRNAs in both constitutive and inducible types.

Antigens, Surface ; analysis ; Base Sequence ; Biological Assay ; Cells, Cultured ; DNA ; analysis ; genetics ; Gene Expression Profiling ; methods ; Humans ; Lipopolysaccharide Receptors ; analysis ; Lymphocyte Antigen 96 ; Membrane Glycoproteins ; analysis ; Molecular Probe Techniques ; Molecular Sequence Data ; Monocytes ; metabolism ; RNA Probes ; analysis ; genetics ; Receptors, Cell Surface ; analysis ; Receptors, Immunologic ; analysis ; Ribonucleases ; Toll-Like Receptor 4 ; Toll-Like Receptors

OBJECTIVETo investigate the effects of tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma) on the expression of pattern recognition receptors (PRRs) on the surface of mouse alveolar macrophages.

METHODSAlveolar macrophages from mouse were cultured in DMEM supplemented with 10% (V/V) endotoxin-free calf serum. After the alveolar macrophages were stimulated with TNF alpha and IFN gamma (concentration, 20 ng/ml) for 3 h, 6 h and 12 h, the expression of PRRs, including cluster of differentiation 14 (CD14), scavenger receptor (SR), toll-like receptor 4 (TLR4), TLR2 and TLR9 mRNA and proteins were examined by RT-PCR and immunohistochemistry.

RESULTSThe expressions of CD14, TLR2 and TLR9 receptors, which were related with cellular activation, were up-regulated by the stimulation of TNF alpha and IFN gamma (P < 0.05), while SR, which was related with cellular defense action, was down-regulated (P < 0.05). Although the expression of TLR4 was up-regulated, there was no statistical significance (P > 0.05).

CONCLUSIONSThe cytokines such as TNF alpha and IFN gamma could also produce feedback regulation on the expression of PRRs at the levels of genes and proteins. Such regulation on the PRRs expression would be significant for further amplification of inflammation cascade and eventually leading to uncontrolled inflammation.

Animals ; Cells, Cultured ; Interferon-gamma ; pharmacology ; Lipopolysaccharide Receptors ; biosynthesis ; genetics ; Macrophages, Alveolar ; metabolism ; Mice ; RNA, Messenger ; genetics ; Receptors, Pattern Recognition ; biosynthesis ; Toll-Like Receptor 2 ; biosynthesis ; genetics ; Toll-Like Receptor 4 ; biosynthesis ; genetics ; Toll-Like Receptor 9 ; biosynthesis ; genetics ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate the polymorphisms of myeloid differentiation-2 (MD-2) gene promoters, and to explore whether such polymorphisms are associated with the susceptibility to multiple organ dysfunction syndrome (MODS) and sepsis in Chinese Han population.

METHODSUsing polymerase chain reaction-restriction fragment length polymorphism method, the authors detected the single nucleotide polymorphisms of the promoter region of MD-2 gene at position - 1625C/G in 105 severe trauma patients (42 with sepsis). The organ function was scored.

RESULTSThe frequency of CC genotype in MD-2 gene promoter region at position - 1625 was 0.5 (21/42) in septic patients and 0.7 (44/63) in non-septic patients. The frequency of CG genotype was 0.38 (16/42) in septic patients and 0.27 (17/63) in non-septic patients. The frequency of GG genotype was 0.12 (5/42) in septic patients and 0.03 (2/63) in non-septic patients. The MODS scores in trauma patients carrying G allele at position - 1625 were significantly higher than those carrying C allele (P<0.001 for dominant effect, and P>0.05 for recessive effect). Moreover, trauma patients carrying G allele appeared to have higher risk of sepsis comparing to those carrying C allele (OR 0.477, 95% CI 0.266-0.855, P<0.05). Sepsis morbidity was significantly different between subjects with C and G alleles (P<0.05 for dominant effect, P>0.05 for recessive effect).

CONCLUSIONSThe polymorphisms of the promoter region of MD-2 gene at position - 1625 C/G is correlated with MODS and sepsis after severe trauma in Chinese Han population. The people with - 1625 G allele in the promoter region of MD-2 gene may be a risk factor of severe complications.

Asian Continental Ancestry Group ; China ; Genetic Predisposition to Disease ; Humans ; Lymphocyte Antigen 96 ; genetics ; Multiple Organ Failure ; etiology ; genetics ; Polymorphism, Genetic ; Promoter Regions, Genetic ; Sepsis ; etiology ; genetics ; Wounds and Injuries ; complications ; genetics

OBJECTIVETo identify the single nucleotide polymorphisms (SNPs) in the regulatory and coding regions of human interleukin-1 receptor type I (IL-1R1) gene and to assess their potential effect on the function of IL-1R1.

METHODSThe 5' flank region, exons, parts of the introns, as well as 3' flank region of IL-1R1 gene were sequenced to identify and characterize the SNPs in Chinese population. Effects of the SNP on the structure and function of IL-1R1 were analyzed by computational methods.

RESULTSSixteen SNPs were identified through a 9643 bp sequencing of IL-1R1 gene. Among them, four were in 5' flank region, four in intron region, one in coding region, and seven in 3' untranslated region. A novel SNP in Chinese population was involved in a structural change in IL-1R1, which may influence the signal transduction of IL-1R1.

CONCLUSIONThe SNP in the IL-1R1 gene might influence its function as an important receptor of IL-1 family.

Amino Acid Sequence ; Asian Continental Ancestry Group ; Cell Membrane ; metabolism ; Computational Biology ; Exons ; genetics ; Humans ; Hydrophobic and Hydrophilic Interactions ; Introns ; genetics ; Molecular Sequence Data ; Polymorphism, Single Nucleotide ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Receptors, Interleukin-1 Type I ; chemistry ; genetics ; metabolism ; Sequence Homology, Amino Acid