Objectives:To evaluate the potent role of matrix metalloproteinases(MMPs) and its tissue inhibitors(TIMPs) in the processes of metastasis and local invasiveness of Chinese human ductal adenocarcinoma of the pancreas,and to evaluate the possible biologic association between its gene expression and the clinical manifestations.Methods:Northern-blot and in-situ hybridization has shown MMPs and TIMPs gene expression in the pancreas and alterations associated with neoplastic transformation.Fifteen cases of surgical pancreatic specimens were examined with cDNA probes to MMP2,MMP9 and TIMP1,findings were correlated with the size of tumor section,CA19-9,pathological classification,thrombosis and infiltration of capsule and lymph nodes.Results:Increased levels of genes mRNA from MMP2,MMP9 and TIMP1 were found in the examined pancreatic cancer tissues,MMP2≈MMP9＜TIMP1,whereas low levels of transcripts for MMP2,MMP9 and TIMP1 were detectable in pancreata of organ donors.Transcripts coding for MMP2,MMP9,TIMP1 were found in both stroma and tumor cells.Gene expression of MMP2,MMP9 and TIMP1 has shown an obvious correlation with the infiltration of capsule cells,surrounding lymph nodes and the specific histopathological features.Conclusions:Imbalance between MMPs and TIMPs may help physicians to assess the metastatic potential and the prognosis of individual patients.

Objective To identify the putative CpG-N ODNs in adenovisus 2 DNA (Adv2 DNA) and Adv5 DNA by comparing the sequence difference among Adv2, 5, 12 DNA and E.coli (EC) DNA. Methods Sequences of Adv2, 5, 12 DNA and EC DNA were obtained from the Entrez Nucleotides database at NCBI. The specific CpG motifs of Adv2 DNA and Adv5 DNA were identified after above sequences were analyzed and compared by softwares such as DNATools, BioEdit, and so on. All the 12-ODNs with specific CpG motif core were searched from Adv2 DNA and Adv5 DNA. Results 19 specific CpG motifs were ascertained and 504 12-ODNs were detected in Adv2 DNA and Adv5 DNA. Conclusion 504 12-ODNs were putative CpG-N ODNs in Adv2 DNA and Adv5 DNA.

Objective To obtain a potent bacterial DNA antagonizing CpG oligonucleotides (CpG-N ODN) from the structures of Adv2, 5 DNA. Methods Ten putative CpG-N ODNs were synthesized and investigated. Their abilities to inhibit the TNF-? release from hPBMC were observed. Based on the above results, the putative CpG-N ODN was redesigned according to the relationship between the structure and free energy using RNA structure software (version 3.71). Eleven putative CpG-N ODNs were synthesized and screened. Results Out of the ten initial CpG ODNs, ODN101 was the only CpG-N ODN with weak activity to inhibit TNF-? release from hPBMC induced by CpG-N ODN. After redesign, five CpG-N ODNs with strong activity were confirmed. Conclusion Six CpG-N ODNs were obtained with activity to inhibit TNF-? release from hPBMC induced by CpG-N ODN.

AIM: To investigate whether the bacterial DNA participates in SIRS and its possible mechanism. METHODS: Escherichia coli genomic DNA (EC DNA) was extracted and purified from Escherichia coli 25922 by alkaline lysis method. Mortality of mice challenged with EC DNA and the changes of TNF-? and IL-6 in rat serum were observed. ANA-1 cells were cultured in vitro, after the cells were stimulated by different concentrations of EC DNA and LPS, the level of TNF-? and IL-6 in supernatant were tested. Meanwhile,expression of TLR9 and TLR4 on cell surface was measured. Activation of NF-?B was also observed. RESULTS: The lethal effect of EC DNA on mice with an obvious dose-effect relationship was observed. The death happened within 24 hours. Calf thymus DNA and DNase I-treated EC DNA did not lead to mice to die. The changes of serum TNF-? and IL-6 in rats induced by EC DNA and LPS were similar, but TNF-? peak level of EC DNA group appeared 1 hour earlier than that of LPS group. In vitro, large amount of TNF-? and IL-6 were released from ANA-1 cells stimulated by EC DNA. High expression of TLR9 and TLR4 was observed on surfaces of THP-1 cells. In particularly, LPS induced strong activation of NF?B. The results suggested other pathway possibly took part in the signal transduction inducea by EC DNA. CONCLUSION: EC DNA has the abilities to lead to death of mice, and induces serum TNF-? and IL-6 level to increase in rats and ANA-1 cells to release cytokines in vitro. High expression of TLR9 and TLR4, strong activation of NF-?B may be its important molecular mechanism, but other pathway probably exists to play an important role.

To evaluate the practical photodynamic therapy (PTD) efficiency on tumor of en-dogenous ALA-induced PP Ⅸ. Methods: Morphoraglcal alterations were observed after PDT 0f protopor-phyrin Ⅸ (PP Ⅸ) by application of endogenous 6-aminolaevulinic acid(ALA) in Kunming mice bearing H22hepatic carcinoma. Results: After PDT, the tumor cells showed degeneration, deformation and nuclearnecrosis and the tumor tissues were defused with hemorrhage and necrosis. The ultrastructural pathologyindicated that mitochondria in tumor cells were injured and destroyed. C0nclusi0n: The pathological alter-ations suggest that PDT of ALA on H22 hepatic carcinoma was conducted by the destruction of mitochon-dria.

Objective To investigate the protective effect of chloroquine on endotoxemia mice and its inhibition on the release of cytokines induced by LPS. Methods A total of 40 mice of Kunming species were randomly divided into four groups: LPS group received LPS at 10 mg/kg, chloroquine group received chloroquine at 20 mg/kg, LPS plus chloroquine group received chloroquine at 20 mg/kg first, then LPS at 10 mg/kg and control group received only 0.9%sodium chloride at 200 ?l/20 g. The mortality was observed within seven days after injection via caudal vein. ANA 1 cell lines were cultivated in vitro . After chloroquine was first added into the cells for 3 hours, the releases of TNF ? and IL 6 in the supernatants induced by different concentrations of LPS were measured. Results Chloroquine could decrease the death of mice due to endotoxin. Mortality dropped from 100% to 50% ( P

Objective To investigate the roles of CpG motifs in the release of cytokines induced by bacterial DNA. Methods Mice were sensitized with injection of D GalN (600 mg/kg) into the abdominal cavity 1 h before experiment. Mortality was observed after purified Escherichia coli DNA(EC DNA), calf thymus DNA(CT DNA), phosphorothioate backbone CpG oligodeoxy nucleotides (CpG ODN) and CG sequence inverted oligodeoxy nucleotides (non CpG ODN) were injected venously into mice. The injection dosage of DNA given was 30 mg/kg but ODN was 10 nmol/mouse. THP 1 cell lines were cultured in vitro . After the above reagents were added into the culture, TNF ?, IL 6, IL 1? levels were detected to observe the different abilities of these reagents to induce the release of cytokines. Results EC DNA and CpG ODN could induce the death of mice and the large amount of release of cytokines, but CT DNA and Non CpG ODN could not. Conclusion CpG ODN has the ability to induce macrophage to release cytokines largely. CpG motifs, which may play an important role in the release of cytokines induced by bacterial DNA, may probably be the structural basis of bacterial DNA.

AIM: To construct a recombinant vector containing gloverin and express it in vitro by Rapid Translation System(RTS500). METHODS: The gloverin cDNA was amplified by PCR and inserted into the prokaryotic expression vector pIVEX2.3. The recombinant product was identified by PCR and enzyme digestion. The positive reconstructed expression plasmid pIVEX2.3-G was expressed by RTS500 in vitro. The expressed protein was identified by SDS-PAGE and western blotting. RESULTS: Positive recombinant plasmid pIVEX2.3-G was successfully constructed. Target protein of 13.8 Kda with 6-his tag was detected by SDS-PAGE and western blotting. CONCLUSION: Gloverin polypeptide is successfully expressed in inactive E.coli in vitro.

Objective To study the physiological indicators and arterial blood gas values,and create a reliable data for researchers using beagle dogs.Methods Beagle were sampled before and after Zoletil anesthesia (10 mg/kg) via intramuscular injection.Then aterial blood gas was detected by blood gas analyzer.Then the physiological indicators,including heart rate,blood pressure and body temperature were measured via large animal ECG.Results For blood gas values,the pH value decreased significantly (P ＜ 0.01).pCO2 increased significantly and pO2 decreased significantly,the differences were significantly (P ＜ 0.05).In contrast,levels of Lac and HCO3-remained unaltered,indicating a typical respiratory acidosis.For physiological indicators,blood pressure increased slightly,but not significant.The heart rate increased significantly (P ＜ 0.01) and body temperature decreased significantly (P ＜ 0.01).There was no difference between sexes.Conclusion Zoletil anesthesia causes respiratory arrest and respiratory congestion,which further induced respiratory acidosis.Meanwhile,anesthesia also results in elevated heart rhythm and lower body temperature.These data indicate that warming and airway clear are necessary during anesthesia in Beagles.Moreover,it should be considered about the effects of anaesthetics when comparing differents in animals before and after treatment.

Objective　To investigate the role of bacterial DNA in systemic inflammatory response syndrome (SIRS). Methods　A total of 100 mice of Kunming species were divided into ten groups: E.coli DNA (30, 20, 10, 5 and 1 mg/kg ), 30 mg/kg of CT DNA, 60Co DNA, DNased DNA, organic residue of DNA extraction and sterile water control. The last two were pre-treated with D-galactoamine (600 mg/kg intra peritoneally). Animals were administratively injected via tail vein. General physical condition and the death rate of mice were observed within 48 h. Results　①Obvious lethal effect of double strand E.coli DNA on mice were observed with a dose-effect correlation, LD50=11.51 mg/kg. ②NO difference in death rate was found in the group of 30 mg/kg E.coli DNA with or without 60Co irradiation (10/10 and 8/10,P>0.05). ③No rats died in the group of DNased DNA, organic residue of DNA extraction and calf thymic DNA (0/10). Conclusion　Bacterial DNA may play an important role in the development of SIRS.