Through intensifying the clinical pathological discussions and clinical pathological dissections,the clinical thinking,iatri- cal responsibility and analytical ability of the medical students have been improved markedly and the abridge function of pathology between basic medicine and clinical medicine has also been reinforced.

[Objective] To observe the clinical efficacy of ZICAOYOUSHA in treating diabetic foot ulcers.[Method] A multi-center ,randomized,double-blind,placebo-control ed study was conducted. A total of 232 patients with diabetic foot ulcers were randomly assigned the treatment group and control group,in foundation treatment at the same time,the therapy group which was treated by External Application ZICAOYOUSHA had 174 patients,the contrast group which was treated by External Application Gentamicin Emery cloth had 58 patients. Observe the aspect improvement situation in two groups separately in accordance with Wagner grading,carry out statistics processing.[Results] Two groups of curative effect indices had significant differ-ence. [Conclusion] ZICAOYOUSHA is an effective drug for external use in treating diabetic foot ulcers.

Objective To compare the analgesia and side-effects of different concentrations of sufentanil combined with 0.125% ropivacaine for postoperative epidural analgesia after general thoracic surgery.Methods Thirty-six ASA Ⅰ-Ⅲ patients (25 males, 11 females) ages 21-64 yrs weighing 42-79 kg undergoing elective general thoracic surgery under general anesthesia were randomly assigned to receive patient-controlled epidural analgesia (PCEA) with 0.125% ropivacaine combined with sufentanil 0.4 (group A, n = 12) , 0.5 (group B, n = 12) or 0.6 ?g?ml-1 (group C, n = 12) . Epidural catheter was placed at T7,8 or T8,9 interspace. The PCEA pump was set up with back ground infusion of 2 ml?h-1 , a3ml bolus dose and a 30-min lock-out period. VAS scores was used to assess analgesia at rest and during movement. The total bolus doses, PCEA button pressing times (effective/actual), vital signs including MAP, HR, respiratory rate, SpO2 and side effects (nausea, vomiting, pruritus and dyspnea) were recorded. Results During the 48 hours after operation the VAS scores in group C were significantly lower than those in group A and B ( P

Objective To introduce the mutated gene coding cysteine into the gene of dsFv VL of human antibody to N terminal fragment of lipopolysaccharide binding protein(LBP)and to express,purify the mutated dsFv VL in bacterium.Methods We reconstructed and sequenced the mutated gene of VL of human mAb Fab to LBP by Mega-primer PCR based on point mutagenesis method.Some codes of FWR1 of VL had been replaced by TGT in order to code cysteine.The DNA sequence of reconstructed VL was inserted into vector pET-28a(+),then VL was expressed by E.coli.BL21 star(DE3)and was purified by chromatography.Finally the activity of VL to bind NH-LBP was determined by ELISA.Results The results showed that the cysteine was introduced into the position 21 amino acid of VL to replace the threonine.The gene of VL was about 650 bp and relative molecular weight of VL was 28?103.VL could bind NH-LBP directly.Conclusion These have laid a foundation for producing the dsFv against NH-LBP.

Objective To construct the recombinant human metapneumovirus(hMPV) (defined as rhMPV NL/1/00 GFP) in vitro by reverse genetics technique. Methods BSR-T7 cells were transfected using LipofectAMINE 2000 with the full-length cDNA plasmid, and four major protein expressing plasmids, pCITE-N, pCITE-P, pCITE-M2.1 and pCITE-L. After 3 d, cells were subjected to one -80℃ freeze-thaw cycle to prepare lysates. The supernatant of lysate was used to inoculate Vero-E6 cells. After 1-4 d, cells were found for the obvious development of cytopathic effects under light microscope and green fluoroscopic signals under fluorescence microscope, and were observed up to 10 d. The supernatant were collected to de-tect virus titer. Viral RNA was extracted from the supernatant and reverse transcriptase polymerase chain re-action (RT-PCR) was used to amplify N, F and G genes of rescued virus. Results Cytopathic effects and green fluoroscopic signals was readily and obviously observed after 1-4 d post-inoculation in Vero-E6 cells, then cytopathic effects got worse and green fluoroscopic signals became stronger gradually up to 10 d. The ti-ters of the 1st, 5th, 10th,15th and 20th generation virus ranged from 105.0 to 106.5 TCID50/ml. Amplicons with size of 910 bp (N), 450 bp (F) and 980 bp (G) by RT-PCR were accordant with expectant. Nucleotide sequence analysis of above cDNA fragments showed 100% similarity with reported sequence of hMPV NI/1/00 strain. The recombinant virus was genetically constant and GFP-labeled after 20 passages in Vero-E6 cells. Conclusion Recombined hMPV was successfully rescued by reverse genetics technique. This study lays ground for exploring pathogenesis of hMPV infection and development of hMPV attenuated vac-cines.

Objective To obtain the disulfide stabilized Fv fragment (dsFv) against N-terminal fragment of human lip polysaccharide binding protein (NH-LBP) and to identify its biological vitality. Methods The disulfide stabilized Fv fragment antibody (dsFv) was obtained after the inclusion bodies of dsFvVH and dsFvVL had been refolded and purified. Then the characteristics of dsFv were determined in vitro by ELISA and by detecting the secretion of tumor necrosis factor alpha (TNF-?) in rats. Results There was 2.1 mg protein of dsFv obtained. dsFv had good combination with NH-LBP and could restrain inflammatory reaction caused by lip polysaccharide (LPS) in vivo. Conclusion It is feasible to get dsFv against NH-LBP by respective expression of VL and VH. The partial inhibition of the biological function of LBP by dsFv is a new way to restrain the over-inflammatory reaction in vivo.

Objective:To investigate the antitumor effect of Actinidia Chinensis Planch polysaccharide(ACPS)in B16-bearing C57BL/6 mice and approach its mechanism.Methods:The C57BL/6 mice model was established by B16 subcutaneous inoculation,giving the polysaccharides from vena caudalis injection,and flow cytometer(FCM)was used to detect the distribution of tumor-cell cycle,and electron microscope was used to survey morphologic transformation about apoptosis.Results:ACPS can inhibit the growth of B16,the high,middle dose groups obviously restrained the tumor with the rate of 48.67%,40.90%and the control group 24.13%,and classical apoptosis corpuscles had been found through electron microscope in ACPS groups.Compared with control group,the ACPS promoted the spleen-index of B16-bearing mice and cut down the proliferation index,and the G1/S phase was at growth-inhibitory concentrations judged by the distributing analysis on cell-cycle.Conclusion:ACPS had obvious effect of restraining B16 and promoting the spleen index of tumor-bearing mice,which maybe due to its function of regulating immunization and the distributing of cell-cycle.

Objective To construct the recombinant adenovirus vector containing human survivin, and transfect it into dendritic cells. Methods Full length survivin cDNA was obtained from recombinant plasmid pCITE-survivin by PCR. The PCR product was double-digested with restriction endonucleases KpnⅠ and XholⅠ, and inserted orientationally into pAdTrack-CMV. The plasmid of pAdTrack-survivin was lined with PmeⅠ, and the fragment containing survivin was reclaimed and transfected into E. coli. BJ5183. After having been screened, the extracted plasmid of positive bacteria was transfected into HEK293 cells with liposome and was identified by the green fluorescence protein (GFP) expression and by PCR method. The virus was transfected into dentritic cells, and the expression of survivin was proved by the GFP expression and Western blot analysis. Results The recombined adenovirus-survivin was constructed successfully and the titer was about 1.65?10 8 pfu/ml. A band was observed by Western boltting and its relative molecular mass was about 16.5?10 3. Conclusion A recombinant adenovirus vector containing human survivin was constructed successfully, and the survivin protein was expressed by dendritic cells.

Objective To reconstruct the proteinic sequence of human cathelicidin LL-37 to increase the bactericidal activity of LL-37 and to express the reconstructed LL-37 (rLL-37) in bacterium. Methods The two dimensional structure, three dimensional structure and chemical characteristic of LL-37 were analyzed by Soft Ware Anthepro 5.0 and SWISS-MODEL. Without the three dimensional changes of LL-37, some negative amino acids of human cathelicidin LL-37 were replaced by positive amino acids and the positive charge of LL-37 was increased. According to the proteinic sequence changes of rLL-37, the DNA sequence of rLL-37 was reconstructed by Touch-Down PCR and recombined with vector pET-28a (+), thus rLL-37 was expressed in E.coli. BL21 (DE3) by the induction of IPTG and was purified by chromatography. Results Glu~ 16 , Asp~ 26 , Glu~ 36 of LL-37 were replaced by Gln~ 16 , Asn~ 26 , Gln~ 36 and the static charge of LL-37 was increased from +5.8 to +9.0 at pH 7.4. The DNA sequence of rLL-37 was reconstructed and inserted into vector pET-28a (+), the rLL-37 was expressed in E.coli. BL21 (DE3) and purified by strong cation exchange supports Macro-Prep High S successfully. The rLL-37 was proved by the means of inhibitory zone to be able to kill Gram-negative bacteria and Gram-positive bacteria. Conclusion It is feasible to reconstruct human cathelicidin LL-37 and express the protein in bacteria by fusion, which make it possible to produce more rLL-37 and study its biological function deeply.