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 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.

Objective To obtain and identify monoclonal antibody against human lipopolysaccharide binding protein (LBP). Methods Three clones of antibody with better activity against human LBP were isolated after human antibody library screening by human LBP and 5 rounds of enrichment. After plasmid extracting, geneⅢ cutting, self-recircling and electric transforming, soluble Fab was expressed in E.coli. XL1-blue by the induction of IPTG and its characteristics were determined. Results The positive antibody was concentrated to 8.16?104 folds and the highest activity of the three clones was 106 . Conclusion Phage antibody against human LBP has been obtained successfully, which lays a solid foundation for researching its function.

Objective To employ 2 approaches to construct and express reconstructed LL-37 (rLL-37) in procaryotic system, and to explore a better preparation method. Methods The first method: the rLL-37 was inserted into vector pET-28a (+), then was induced to express in E.coli. BL21 (DE3) and purified by chromatography; the second method: the rare codons in the rLL-37 gene sequence were substituted by the preferred codons of procaryotic cell, and a fragment of carrier protein molecule (CPM) was added to the N termination of the objective sequence to construct expression plasmid pET-30a(+)-CPM-rLL-37, then the rLL-37 was expressed in E.coli. BL21 Star(DE3) and purified by chromatography. The productive rates of the 2 methods were compared and the antimicrobial effects of obtained rLL-37 was studied. Results The first method: the DNA sequence of rLL-37 was obtained successively by Touch-Down PCR. The expression plasmid pET-30a(+)-CPM-rLL-37 was expressed with fusion protein in E.coli BL21 (DE3). The expression rate accounted for 20% of total bacterio-protein, then the expressed product was purified by using high positive ion exchange column Macro-Prep High S; The second method: a fragment of carrier protein molecule was designed that contained 28 amino-acid residue and its pHi was 2.7, net charge was-6.0 at pH 7.4. After the expression plasmid pET-30a(+)-CPM-rLL-37 was constructed successively, it was expressed in E.coli BL21 Star (DE3). The expressed fusion protein accounted for 35% of total bacterio-protein, then the expressed product was purified by using affinity binding chromatography with TALON resins successfully. The obtained 2 kinds of rLL-37 were able to kill both Gram-negative and-positive bacteria by the means of inhibitory zone. Conclusion It’s feasible to prepare efficiently rLL-37 in procaryotic system, which founds the basis for the further research on bactericidal activity of rLL-37.

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 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 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.