Zheng-Guo Wang

The AMPA type of glutamate receptors (AMPARs)-mediated excitotoxicity is involved in the secondary neuronal death following traumatic brain injury (TBI). But the underlying cellular and molecular mechanisms remain unclear. In this study, the role of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in GluR2-lacking AMPARs mediated neuronal death was investigated through an in vitro stretch injury model of neurons. It was indicated that both the mRNA and protein levels of PTEN were increased in cultured hippocampal neurons after stretch injury, which was associated with the decreasing expression of GluR2 subunits on the surface of neuronal membrane. Inhibition of PTEN activity by its inhibitor can promote the survival of neurons through preventing reduction of GluR2 on membrane. Moreover, the effect of inhibiting GluR2-lacking AMPARs was similar to PTEN suppression-mediated neuroprotective effect in stretch injury-induced neuronal death. Further evidence identified that the total GluR2 protein of neurons was not changed in all groups. So inhibition of PTEN or blockage of GluR2-lacking AMPARs may attenuate the death of hippocampal neurons post injury through decreasing the translocation of GluR2 subunit on the membrane effectively.

To 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. Using 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. The 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 (P0.05 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, P0.05 for recessive effect). The 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.

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

To explore the pathomorphological changes in abdominal viscerae injuries produced by underwater blast waves. Sixty-one adult mongrel dogs were used in this study. Underwater explosions in different intensity were produced by detonating 200 g, 500 g and 1,000 g TNT, respectively. The physical parameters of blast wave were measured by PCB dynamic pressure transducers specially designed for underwater explosion. The pathomorphological changes in abdominal viscerae were observed at 6 hours after injury. The physical features of underwater blast wave were characterized by high peak pressure, short positive action duration, and higher intensity of impulse. The pathomorphological observation revealed high incidence of injuries of gastrointestinal tract (62.30 percent), manifesting mainly various degrees of subserosal and submucous bleeding in gastrointestinal tract. Rupture of seromuscular layer, subserosal hematoma, and even, perforations of gastrointestinal tract occurred in some animals. Injuries of the liver, spleen, pancreas and other parenchymatous organs injuries, such as subcapsular bleeding, hematoma, and ruptures, appeared with low incidence. No obvious injuries were found in the gallbladder or urinary bladder and other fluid containing organs. Underwater blast wave can induce injuries to many abdominal viscerae. Gastrointestinal tract injuries, occurring in high incidence are usually severe, and they should be given early treatment in underwater blast injuries.

Early 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. Middle 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. The 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. Instead 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.

To investigate the effect of substance P (SP) on gene expression of transforming growth factor beta-1 (TGFbeta-1), transforming growth factor receptor-1 (TGFR-1) and transforming growth factor receptor-2 (TGFR-2) in fibroblasts cultured in vitro from rat's granulation tissues. The fibroblasts from the granulation tissues in the skeletal muscle of rat's hind limbs injured by formaldehyde were cultured in vitro. When different concentrations (10(-9)-10(-5) mol/L) of SP were added into the culture medium, the changes of gene expression of TGFbeta-1, TGFR-1 and TGFR-2 in the cultured fibroblasts were observed with reverse transcription polymerase chain reaction at different intervals (0, 3, 6, 12 and 24 hours after incubation). The gene expression of TGFbeta-1, TGFR-1 and TGFR-2 in the fibroblasts cultured from rat's granulation tissues was up-regulated by SP. The peak level of the mRNA expression was found at 10(-8) mol/L SP and the up-regulation effect was not found at 10(-5) mol/L and 10(-6) mol/L. The peak levels of gene expression of TGFbeta-1, TGFR-1 and TGFR-2 in the fibroblasts treated with SP were achieved at 6 and 12 hours, respectively. SP has up-regulation effect on the gene expression of TGFbeta-1, TGFR-1 and TGFR-2 in fibroblasts from rat's granulation tissues in vitro, and the effect is related to different stimulating concentrations of SP. It may be concerned with proliferation and differentiation of fibroblasts and formation of scar tissues during wound healing.

To study the modulating effect of sensory neuropeptide substance P (SP) on gene expression of epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2) and EGF receptor (EGFR), FGF receptor-1 (FGFR-1) in granulation fibroblast. The study consisted of two parts, in vivo and in vitro. In vivo, fifty wistar rats were randomly arranged into capsaicin group and control group. The rats in capsaicin group were subcutaneously injected with neurotoxin dose of capsaicin (50 mg/kg) on the back of the rats to chemically destroy sensory nerves to prevent the release of SP. After one week, full-thickness skin wounds were created on the rats' back. SP content and EGF, EGFR, FGF-2, FGFR-1 gene expression in the granulation tissue were observed using immunohistochemistry and in situ hybridization combined with image analysis on 3rd, 6th, 9th 12th after skin injury, respectively. The experimental procedure in control group were similar with that in capsaicin group except injection with capsaicin. In vitro, SP (10(-9)-10(-5) mol/L) was added into culture medium for cultured fibroblasts from granulation and mRNA expressions of EGF, FGF-2 and their receptors were assayed with RT-PCR. In the present study, immunoreactive stain for SP in granulation tissue were correlated with gene expression of EGF, FGF-2 and their receptors using in situ hybridization in vivo. Once sensory nerves were destroyed and SP release was inhibited in capsaicin group, the gene expression of above growth factors and their receptors were attenuated. SP concentrations in culture medium for upregulation of mRNA expressions of EGF and EGFR in cultured fibroblasts were from 10(-8) mol/L to 10(-6) mol/L and from 10(-6) mol/L to 10(-5) mol/L, respectively. SP concentration was from 10(-9) mol/L to 10(-5) mol/L for upregulation of FGF-2 mRNA expression in cultured fibroblasts and from 10(-6) mol/L to 10(-5) mol/L for upregulation of FGFR-1. Sensory neuropeptide SP released from sensory nerve participates in upregulation of gene expressions of EGF, EGF-2 and their receptors in granulation fibroblasts during wound healing.

To 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. The 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. The 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. These 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.

To investigate the changes of scavenger receptor (SR) and CD14 in Kupffer cells in endotoxemia in order to uncover the mechanism of the liver to turn a defense organ into effector one in sepsis. Mouse models of endotoxemia of different severity were reproduced by injection of different doses of lipopolysaccharide (LPS) via the tail vein. The expression of SR and CD14 in the liver was assayed by immunohistochemistry and was subsequently analyzed with an image analysis system. The levels of TNF-alpha and IL-6 in liver tissue were determined with ELISA. The expression of SR in the liver in the high-dose group was markedly decreased one hour after injection of LPS, and also in the low-dose group at 3 hours. The expression of SR in the liver in the two groups was shown to be progressively decreased with the time prolonged. There was significant difference in average optical density (OD) values of SR between the two groups. The expression of CD14 in the two groups was shown to be significantly increased one hour after injection of LPS, and more significantly with the time prolonged. But there was no significant difference in OD values of CD14 between the two groups. The contents of intrahepatic proinflammatory mediators TNF-alpha, IL-6, ALT and TBIL were significantly increased after injection of LPS. Correlation analysis revealed that the changes of TNF-alpha, IL-6, ALT, and TBIL were negatively correlated with the expression of SR, and positively with the expression of CD14. The up-regulation of CD14 expression and down-regulation of SR expression on Kupffer cells might be one of the important mechanisms for the conversion of Kupffer cells from immune defensive to inflammatory response cells in acute hepatic injury.