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 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.

BACKGROUND:Adult cardiomyocytes show no regenerative ability, and celltherapy for myocardial regeneration and repair may improve myocardial ischemic injury function. OBJECTIVE:To confirm the effect and reveal the mechanism of bone marrow mesenchymal stem cells (BMSCs) on acute myocardial infarction (AMI). METHODS:BMSCs were isolated, cultured from bone marrow of Sprague-Dawley rats using density gradient centrifugation. AMI models were produced in 20 rats by ligating the left anterior descending (LAD) coronary artery, and randomly divided into model group and BMSCs group. In the BMSCs group, cells were subsequently injected with a sterile microinjection via the tail vein. RESULTS AND CONCLUSION:Six months postoperatively, the cardiac function was improved, the vessel density was increased, the percentage of apoptotic cells was decreased in the BMSCs group than that in the model group;the expression levels of inflammatory factors, including vascular endothelial growth factor, von Wil ebrand factor, transforming growth factor 3β, and interleukin-1βmRNA were significantly improved in the BMSCs group than that in the model group. These results showed that BMSCs can protect the myocardium from AMI by regulating the secretion of inflammatory cytokines and angiogenic factors.

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 compare the pharmacokinetics and relative bioavailability between matrine sustained release tablet,matrine conventional capsule and matrine injection. Methods: Dogs were given single oral dose and multiple doses of matrine sustained release tablet, capsule or injection in a randomized crossover way. Matrine concentrations in dog plasma were determined by RP-HPLC method. Results: The results showed that the t max and c max were 300 min and (5.088?0.490) ?g/ml for matrine sustained release tablet, (85?12) min and (6.360?0.215) ?g/ml for the conventional capsule, and 10 min and (6.500?0.404) ?g/ml for the injection. The relative bioavailability of the sustained release tablet was (153.7?9.4)% compared with that of the conventional capsule; the absolute bioavailability of the sustained release tablet was (73.5?14.2)% compared with that of the injecetion. The in vivo absorption rate of the sustained release tablet was significantly correlated with the in vitro release rate(r=0.981 2,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 investigate the clinical application value of stathmin, p16 and Ki-67 in the cervical intractable cases. Methods Immunohistochemical method was used to detect the expressions of stathmin, p16 and Ki-67 in surgical specimens of 288 cervical intractable cases, including 30 cases of cervical benign changes, 70 cases of cervical intraepithelial neoplasia (CIN)Ⅰ, 78 cases of CINⅡ, 85 cases of CINⅢand 25 cases of squamous cell carcinoma (SCC, as control group). The application value of stathmin, p16 and Ki-67 in the cervical cases were analyzed. Results The positive expression rates of Ki-67 of cervical benign changes and CINⅠwere 20.0 % (6/30) and 54.3 % (38/70) (χ2 = 3.29, P> 0.05). The expression rates of Ki-67 in CINⅡ, CINⅢ and SCC were all 100.0 %, and compared with the cervix benign changes, the differences were statistically significant (χ2= 112, P< 0.05). The expression rates of p16 in cervical benign changes and CINⅠwere 6.7 % (2/30) and 91.4 % (64/70), and there was significantly statistical difference (χ2=50.64, P<0.05). However, the expression rates of p16 in CINⅡ, CINⅢand SCC were all 100.0%, and compared with the cervix benign changes, the differences were statistically significant (χ2= 7.18, P< 0.01). The expression rates of stathmin in cervical benign changes, CINⅠ, CIN Ⅱ, CINⅢ and SCC were 3.3 %(1/30), 5.7 % (4/70), 23.1 % (18/78), 77.0 % (67/87) and 100.0 % (25/25), respectively, and there was no statistic difference in cervical benign changes, CINⅠand CINⅡ (χ2=0.68, P>0.05), but the expression rates in CINⅢ and SCC were higher than those in cervical benign change, CINⅠand CIN Ⅱ(P< 0.01). The positive expressions of stathmin, p16 and Ki-67 in each group of CIN were positively correlated (r= 0.412, P< 0.05). Conclusions Combined detection of p16 and Ki-67 can assist in the differential diagnosis of cervical intractable cases, and provide objective indicators for the classification and accurate diagnosis of CIN. Combined detection of p16 and stathmin may help to identify high-grade, low-grade CIN and cervix benign changes for the reduction of over-treatment.

Objective To explore the effects of comprehensive rehabilitation on patients with stable chronic obstructive pulmonary disease (COPD) in rural communities.Methods Between June 2010 and June 2012,212 stable COPD patients were randomly divided into management group (n =107) and control group (n =105).All patients received conveutional treatment.In addition,107 stable COPD patients relying on new rural cooperative medical service station in management group underwent community comprehensive rehabilitation including training in respiratory function,exercise training,nutrition intervention and psychological care.Effect of lung function,symptoms (times of acute attack & hospitalization) and quality-of-life (QOL) rated by SF-36 questionnaire between two groups were recorded at Month 6.Results The score changes of QOL had significant inter-group differences between 2 groups for PCS (scores for physical component summary) and MCS (scores for mental component summary) (11.4 ±8.2 vs.1.6 ± 1.2,5.5 ±3.5 vs.2.2 ±0.9,all P ＜0.01).In the scores for physical component summary,the score change of physical function,physiological function,body pain and general health were significantly different between two groups (6.7 ±4.3 vs.1.2 ±0.8,10.9 ±6.3 vs.1.9 ± 1.5,6.4 ±4.7 vs.3.6 ±2.7,3.2 ±2.7 vs.1.6 ± 1.1,P ＜ 0.01).In the scores for mental component summary,the score change of vitality,emotional function,social function and mental health were significantly different between two groups (4.9±3.2 vs.1.9±1.4,2.7±2.1 vs.1.6±1.1,11.6 ±9.2 vs.3.6 ±2.3,6.7 ±4.3 vs.1.4±0.9,P＜0.01).The times of acute attack and hospitalization were obviously lower than those of the control group.No significant inter-group difference existed in lung function.Conclusion Comprehensive rehabilitation may improve the QOL in stable COPD patients in rural communities and reduce their times of acute attack.

Objective To evaluate the effectiveness of management for patients with chronic obstructive pulmonary disease (COPD) in stage Ⅰ at new rural cooperative medical facilities.Methods A quasi-experiment design was used for the evaluation of patients registering at Dongcun and Fangyuan subdistrict offices in Haiyang.And 79 COPD cases from Dongcun sub-district offices were selected into the management group while 76 cases from Fangyuan the control group.The measures at the new rural cooperative medical facilities included smoking-free publicity,avoiding a hazardous environment,application of influenza vaccines and training of patients and local general practitioners in essences of COPD prevention and treatment.Except for routine treatment,no special measures were taken for the control group.ResultsSixty-one patients in the management group and 60 in the control group completed a 3-year follow-up.Fifteen patients were smokers during their initial visits in the management group and 13 quitted smoking after a 3-year management.Meanwhile 19 patients were smokers during their initial visits in the control group and 3 of them quitted smoking within the same period.The decrement of BODE index between post-management at year 2&3 and between pre-management was higher in the management group versus the control group within the same period (P ＜ 0.01).The annual average frequencies of common cold,acute attack and physician consultation were obviously lower post-management at year 3 than those pre-management and the control group within the same period ( P ＜ 0.05 ).Lung functions of two groups deteriorated as compared with those 3 years before ( P ＜ 0.05).But no statistical difference existed between two groups (P ＞ 0.05).ConclusionThe management of COPD patients in stage Ⅰ at new rural cooperative medical facilities can improve their quality of life and reduce their frequencies of common cold,acute attack and physician consultation.