In order to explore the relationship between xenograft rejection and cytokines, the proliferative responses and cytokine contents from human lymphocyte were detected in porcinehuman mixed lymphocyte culture (MLC) and compared with human allo-MLC. The results showed that the proliferative responses in the former was equal to or stronger than that in the latter, and the kinetics of IL-2 and IFN-Y in both supernatants were similar but the peak of IL-4 from the former was higher than that from the latter (P<0.05). This suggests that cytokines are likely to play roles in porcine-human MLC.

Objective To construct a prokaryotic expression system for expressing the phosphatase-encoding gene phpP in StkP/PhpP signaling couple in Streptococcus pneumonia ( S.pneumoniae) strains, and to further understand the phosphatase activity of the recombinant protein rPhpP.Methods The entire phpP gene of S.pneumoniae strain ATCC6306 was amplified by PCR.The PCR products were sequenced.A prokaryotic ex-pression system for expressing the phpP gene was constructed by the genetic engineering technique.The ex-pressed protein rPhpP and the solubility of rPhpP were assessed by SDS-PAGE and gel image analyzer.Ni-NTA affinity chromatography was performed to purify rPhpP.The changes of phpP gene transcription after the treat-ment with sublethal dosages of penicillin and cefotaxime were determined by real-time fluorescent quantitative RT-PCR.The functional domain in the sequence of the phpP gene and its type was analyzed by bioinformatic softwares.The activity of rPhpP in hydrolyzing the substrate of PP2C phosphotase was measured with Serine/Threonine Phosphotase Assay Kit.The enzyme kinetic parameters of rPhpP were calculated.Results The se-quence of the cloned phpP gene was identical with that reported in GenBank.The rPhpP in soluble form was ex-pressed in the constructed prokaryotic expression system.An increased expression of phpP gene at mRNA level was induced by sublethal dosage of penicillin or cefotaxime.The domain of PP2Cc type phosphatase was detec-ted in the sequence of phpP gene.The purified rPhpP protein could hydrolyze phosphopeptides [ RRA ( pT) VA], a substrate of PP2C type phosphatase, in a dose-dependent manner with Km and Kcat values of 277.35μmol/L and 0.71 S-1 ,respectively.Conclusion The protein encoded by phpP gene of S.pneumoniae was a PP2C type phosphatase.The expression of phpP gene could be enhanced by sublethal dosage of penicillin or ce-fotaxime.

Objective To construct a mutant strain of Streptococcus pneumoniae ( S.pneumoniae) with ciaH gene-knockout (ΔciaH) and to analyze the correlation between the ciaH gene and the bacterial re-sistance against β-lactam antibiotics.Methods The ciaH gene segament of S.pneumoniae strain ATCC6306 was amplified by PCR.The PCR product was sequenced after T-A cloning.A suicide plasmid pEVP3ciaH was constructed for the deletion of ciaH gene and then transformed into the ATCC 6306 strain by using the CaCl2 method .The mutant strain of S.pneumonia strain ATCC6306 with ciaH gene-knockout (ΔciaH) was genera-ted through homologous recombination , insertion inactivation and amphemycin screening , which was further identified by PCR , sequencing analysis and laser confocal microscopy .Double agar dilution method was used to detect the minimal inhibitory concentrations ( MICs ) of penicillin G ( PCN ) and cefotaxime ( CTX ) against theΔciaH mutant strain and the wild type strain .The differences between the MICs were further ana-lyzed.The changes of ciaH gene expression at mRNA level after treatment with 1/4 MIC of PCN or CTX were detected by real-time fluorescent quantitative RT-PCR ( qRT-PCR ) .Results The ciaH gene in the genomic DNA of the generated ΔciaH mutant strain was inactivated by insertion as indicated by PCR and se-quencing analysis .Results of the immunofluorescence assay showed that the ΔciaH mutant strain did not ex-press the CiaH protein .The MICs of PCN and CTX against the ΔciaH mutant strain were 32 μg/ml and 64μg/ml, respectively, which were significantly higher than that of the wild type strain (0.06 μg/ml and 1μg/ml) (P<0.01).The expression of ciaH gene at mRNA level was significantly elevated after treatment S.pneumoniae ATCC6306 strain with 1/4 MIC PCN or CTX (P<0.01).Conclusion The CiaH protein in the CiaH/CiaR two-component signaling system is involved in the resistance of S.pneumoniae against β-lac-tam antibiotics.

BACKGROUND: Existing research shows that in situ regeneration of skin deep within the second degree bum wound and donor site wound healed without physical scarring, can promote three-degree burn wounds liquefied necrotic tissue removement, the growth of transplanted skin, reduce scar; scar-shift using the in situ regeneration is expected to reach significantly reduce scar symptoms, and to reduce the effect of scar, which have not be reported.OBJECTIVE: To observe effects of skin regeneration in situ method to remove scar in the treatment of hypertrophic scar. METHODS: A total of 32 patients with many hyperplastic scars, including 25 males and 7 females, aged 16-52 years, disease course of 1-11 years. Two similar scar regions were selected from each patient for self control. In the experimental group, scar removal, scar skin replantation after the application of in situ regeneration of the skin treatment using burn cream coated yarn. In the control group, scar removal, scar skin replantation after the application of traditional Vaseline covered by treatment. Curative effects were observed and compared. Scar hyperplasia was assessed using Vancouver Scar Assessment Scale assessment. RESULTS AND CONCLUSION: Replanted scar skin explants were survived in both groups. In the experimental group, healing speed and quality of wound surface were better than the control group (P< 0.05). After 6 months, the Vancouver Scar Assessment Scale assessment in the experimental group was better than control group (P < 0.05, P < 0.01). Scar caused by pain, itching and other symptoms disappeared, skin formation and color back to pre-implantation were significantly improved compared with the surrounding skin almost. Results indicated that with regarding to the lack of autologous skin source, large area of scar in patients with hypertrophic scars or unwilling to add a new donor site wounds in patients, in situ replantation method is an ideal approach.

Objective: To observe the efficacy of skin regenerative medical technique in treating proliferative scars. Method: Select 32 patients (age16-52) with proliferative scars after burns or wound for 1-11 years,which include 25(male) and 7(female). 2 scar similar spots are chosen in each patient for self-comparison.After the experimental group uses the scar detachment, scar Pi Huizhi applies the beautiful valuable moist burn medicinal plaster gauze cover the cooperation of Chinese and Western medicine home position skin regenerative method treatment; After the control group uses the scar detachment, scar Pi Huizhi applies the petroleum jelly cover the traditional method treatment.The observation comparison curative effect, applies the Vancouver scar appraisal meter appraisal scar proliferation situation. Results: Two groups return to the scar skin which plants to survive.The experimental group regenerates the skin to be good, the cicatrization speed and the quality surpass the control group (P

Objective To study the effects of CO2 pneumope ritoneum on the abdominal viscera metastasis and peritoneal implantation of gastric cancer cell line MKN45 during laparoscopic gastric surgery.Methods The gastric cancer cells metastatic model was eslhblished by injecting 1 ml(2×107/ml)human gastric cancer cell line MKN45 susDension into the caecum wall of 60 Balb/C mice.Two weeks later,the Balb/C mice were divided into CO2 pneumoperitoneum group(n=30)and laparotomy group(n=30). All mice were sacrificed 7 weeks later to investigate the abdominal viscera metastasis and peritoneal implantation of gastric cancer cells.Results Twenty-seven rats in CO2 pneumoperitoneum group were induced with tumor,including 8 with troear port implantation and 19 with organ metastasis;26 rats in laparotomy group were induced with tumor,including 7 with tracar port implantation and 19 with abdominal viscera metastasis,and the differences between the two groups had no statistical significance(x2=0.007,0.240,0.202,0.106,0.042,P＞0.05).Conclusions CO2 pneumoperitoneum does not stimulate the metastasis of gastric cancer(cells to peritoneum and abdominal viscera in Balb/C mice.Laparoscopic surgery for gastric cancer is safe and feasible.

Objective To construct a ciaR gene-knockout (ΔciaR) mutant of Streptococcus pneu-moniae ( S. pneumoniae) and to investigate the effects of CiaR in CiaH/CiaR, a streptococcal two-component signal-transducing system, on the expression of genes encoding penicillin-binding proteins ( pbps genes) and cia-dependent small RNAs (csRNAs). Methods Electrophoretic mobility shift assay (ESMA) was per-formed to detect the recombinant CiaR (rCiaR)-binding pbps genes. A suicide plasmid pEVP3ciaR for ciaR gene knockout was constructed and then aΔciaR mutant was obtained through homologous recombination and insertion inactivation of the suicide plasmid, and screening with chloromycin. The mutant was identified using PCR and sequencing analysis. E-test was used to detect the minimal inhibitory concentrations ( MIC) of penicillin ( PCN) and cefotaxime ( CTX) against S. pneumoniae strains. Changes and differences in the expression of pbps genes and csRNAs in theΔciaR mutant and its wild-type strain before and after treatment with 1/4 MIC of PCN or CTX were detected using real-time quantitative RT-PCR. Results The rCiaR could bind to the promoter regions in pbp1a, pbp1b and pbp2b genes of S. pneumoniae. The ciaR gene in ΔciaR mutant was inactivated by insertion according to the results of PCR and sequencing analysis. After treatment with 1/4 MIC of PCN or CTX, the expression of pbps genes at mRNA level ( pbps-mRNAs) in theΔciaR mu-tant was significantly increased (P<0. 05), but the levels of csRNAs were significantly decreased (P<0. 05);whereas a significantly decreased pbps-mRNAs (P<0. 05) and increased csRNAs (P<0. 05) were observed in its wild-type strain. The result of E-test showed that the MICs of PCN and CTX against ΔciaR mutant were increased by 250-fold as compared with those against its wild-type strain. Conclusion The CiaR can enhance the drug resistance of S. pneumoniae to PCN and CTX through down-regulating the expres-sion of PBP1a, PBP1b and PBP2b and up-regulating the expression of csRNAs to inhibit the expression of PBPs.

ObjectiveTo generate the prokaryotic expression systems of vwA1 and vwA2 genes of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai,and to determine the correlation among the target recombinant expressed products rVwA1 and rVwA2 and platelet-associated hemorrhage.MethodsThe vwA1 and vwA2 genes of L.interrogans strain Lai were amplified using high fertility PCRs and then the amplification products were sequenced.The prokaryotic expression systems of vwA1 and vwA2 genes were generated by using routine genetic techniques.The expression and dissolubility of rVwA1 and rVwA2 proteins were examined by SDS-PAGE plus Bio-Rad Gel Image Analyzer.Ni-NTA affinity chromatography was used to extract the expressed rVwA1 and rVwA2.Real-time fluorescence quantitative RT-PCRs were performed to determine the changes of vwA1-mRNA and vwA2-mRNA levels in the leptospires of L.interrogans strain Lai before and after infection of human umbilical vein endothelial cell line (HUVEC).The ability of leptospiral rVwA1 binding to human platelets was detected by flow cytometry.A hydrolytic test plus SDS-PAGE were applied to examine the cleavage of leptospiral rVwA2 by a human von Willebrand factor lysase (ADAMTS13).ResultsThe nucleotide and putative amino acid sequences of the cloned leptospiral vwA1 and vwA2 genes were 100％ identities compared to the reported corresponding genes.The generated prokaryotic expression systems of vwA1 and vwA2 genes could express soluble rVwA1 and rVwA2,respectively.When L.interrogans strain Lai infected HUVEC for 8 h,both the vwA1-mRNA and vwA2-mRNA levels were significantly up-regulated (P＜0.05).The result of flow cytometry showed that the leptospiral rVwA1 was able to combine with human platelets with a 60.8％ binding rate. Human recombinant vWF-A2 ( rhvWF - A 2 ),but not the leptospiral rVwA 2,could be hydrolyzed by human ADAMTS 13.Conclusion The vwA1 and vwA2 genes may play roles during infection of L.interrogans species,and the function of vwA1 gene product is referring to the hemorrhage in leptospirosis.

SELEX is a newly developed biochemical technique,which filter out high specificity and high affinity ligand for the target molecules through the identification of aptamer combined with the target molecules.The specific aptamer was used in a variety of clinical applications,such as diagnosis of the disease,development of new therapeutic drugs and even directly applied to disease treatment.

Objective To investigate the correlation between Streptococcus pneumoniae (S.pneumoniae) StkP kinase and drug resistance and to analyze the binding ability of StkP extracellular region (EC-StkP) to β-lactam antibiotics.Methods A stkP gene knockout (ΔstkP) mutant was constructed from S.pneumoniae strain ATCC6306 by insertional inactivation method.E-test was performed to detect the minimum inhibitory concentrations (MIC) of penicillin (PCN) and cefotaxime (CTX) against ΔstkP mutant and its wild-type strain.Bioinformatic softwares were used to predict the EC-StkP of S.pneumonia strain ATCC6306,to generate the three-dimensional structure model of EC-StkP and to analyze the correlation between the structure and functions of EC-StkP.PCR was performed to amplify the extracellular segment of stkP (EC-stkP) gene and the product of it was sequenced after T-A cloning.A prokaryotic expression system of EC-stkP gene was constructed.SDS-PAGE in combination with a gel image analysis system was used to detect the expression of the recombinant EC-StkP (EC-rStkP).The expressed EC-rStkP was extracted by Ni-NTA affinity chromatography.The binding abilities of EC-rStkP to PCN and CTX were detected by isothermal titration calorimetry (VT-ITC) and surface plasmon resonance (Biacore).Results S.pneumonia strain ATCC6306 was sensitive to PCN (MIC=0.06 μg/ml) and CTX (MIC=0.12 μg/ml),but its ΔstkP mutant was resistant to the two antibiotics (PCN MIC=16 μg/ml,CTX MIC=32 μg/ml).The 295 aa segment was predicted as the extracellular region at C-end of StkP of S.pneumoniae strain ATCC6306,containing four penicillin-binding proteins and Ser/Thr kinase-associated (PASTA) domains.The cloned EC-stkP segment and the EC-stkP segment in GenBank shared 99.6% similarity in nucleotide sequence and 100% in amino acid sequence.The constructed prokaryotic expression system for EC-stkP gene expressed EC-rStkP in soluble form.Both PCN and CTX could bind to EC-rStkP and CTX was better than PCN in term of binding ability.Conclusion The stkP gene of S.pneumonia is closely related to drug resistance and the encoded protein,Ser/Thr kinase StkP,can recognize and bind to β-lactam antibiotics.