Objective To construct eukaryotic expression vector of human antimicrobial peptide LL-37 pPIC9-LL-37, and transform the plasmid pPIC9-LL-37 into P.pastoris GS115 to obtain the recombinant P.pastoris strains.Methods The full-length of antimicsobial peptide LL-37 gene was artificially synthesized by overlap extension method and was fused to pPIC9 and then the fused plasmid was transformed into E.coli DH5?.After analysis by PCR and sequencing,the plasmid pPIC9-LL-37 was transformed into P.pastoris.The colonies exhibiting the phenotype of His+Mut+ or His+Mut-were screened by means of MM and MD plates and the insertion was confirmed by PCR.Results The results of PCR and sequencing confirmed that the LL-37 gene was correctly inserted into pPIC9. The colonies of 10 His+Mut+ and 9 His+Mut-were obtained by means of MM and MD plates screening and were confirmed by PCR.Conclsion The recombinant P.pastoris strains containing LL-37 were successfully obtained.

Objective To evaluate the effects of sevoflurane on hippocampal neurogenesis in den-tate gyrus (DG) of mice of different ages. Methods Ninety-six SPF healthy male C57BL∕6 mice, aged 2 weeks, 6 weeks, 9 months and 20 months (24 mice for each age, 12 mice for each group), were divided into 2 groups (n＝48 each) using a random number table method: control group (group C) and sevoflurane group (group S). Group S inhaled 3. 0% sevoflurane for 2 h once a day for 3 consecutive days, while group C inhaled the mixture of air and O2. Six mice of each age were selected, and 5′-bromo-2′-deoxyuridine (BrdU) 50 mg∕kg was intraperitoneally injected immediately before and after inhalation once a day for 3 consecutive days in two groups. Mice were sacrificed at 24 h after the last inhalation (T1), brains were re-moved and hippocampi isolated for determination of the number of nestin and doublecortin ( DCX) positive cells in DG by immunohistochemistry. Mice were sacrificed at 4 weeks after the last inhalation ( T2), brains were removed and hippocampi isolated for determination of the number of neuronal nuclei antigen (NeuN)∕BrdU and glial fibrillary acid protein ( GFAP )∕BrdU positive cells by immunofluorescence. Re-sults Compared with group C, the number of nestin and DCX positive cells was significantly reduced at T1, and the number of NeuN∕BrdU and GFAP∕BrdU positive cells was reduced at T2in mice of 2 weeks and 20 months old (P＜0. 05), and no significant change was found in the indices mentioned above in mice of 6 weeks and 9 months old in group S ( P＞0. 05). Conclusion Three percent sevoflurane can inhibit hipp-ocampal neurogenesis in DG of immature and old mice and exerts no influence on hippocampal neurogenesis in DG of juvenile and adult mice.

Objective To evaluate the role of autophagy in cognitive decline caused by sevoflurane anesthesia and the relationship with neurogenesis in aged mice.Methods Forty-five healthy SPF male mice,aged 20-22 months,weighing 25-35 g,were divided into 3 groups (n=15 each) using a random number table method:control group (group C),sevoflurane anesthesia group (group S) and autophagy agonist rapamycin group (group R).Rapamycin 0.2 mg/kg was intraperitoneally injected every day for 7 days in group R,while the equal volume of solvent dimethyl sulfoxide was given instead in S and C groups.In group S and group R,3％ sevoflurane was inhaled for 2 h once a day for 3 consecutive days starting from 5th day of administration,while the mixture of air and oxygen was inhaled instead in group C.Five mice in each group were randomly selected after the last anaesthesia and sacrificed,and the hippocampus was removed for determination of the expression of microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) and Beclin-1 by Western blot.The other mice were sacrificed after Morris water maze test was performed,and hippocampi were isolated for determination of doublecortin (DCX) positive cells in the dentate gyrus by immunohistochemistry.Results Compared with group C,the escape latency was significantly prolonged,the percentage of time spent in the target quadrant was decreased,the expression of LC3 Ⅱ and Beclin-1 was down-regulated,LC3 Ⅱ/LC3 Ⅰ ratio was decreased,and DCX positive cell counts were reduced in S and R groups (P＜0.05).Compared with group S,the escape latency was significantly shortened,the percentage of time spent in the target quadrant was increased,the expression of LC3 Ⅱ and Beclin-1 was up-regulated,LC3Ⅱ/LC3 Ⅰ ratio was increased,and DCX positive cell counts were increased in group R (P＜0.05).Conclusion Autophagy is involved in the process of cognitive decline caused by sevoflurane anesthesia,which is related to inhibiting neurogenesis in the hippocampus of aged mice.

Objective:To explore the application value of bedside lung ultrasound score(LUS)in evaluating patients with primary graft dysfunction(PGD)after lung transplantation.Methods:A total of 35 PGD patients after lung transplantation in surgical intensive care unit from June 2018 to May 2020 were selected as research objects. After lung transplantation, one physician collected the clinical data, including age, gender, preoperative 24 h APACHE II score, postoperative hemodynamic parameters, lactate and respiratory parameters; another physician was responsible for LUS examination and LUS at 12/24/48/72 h post-operation. The correlation between LUS and oxygenation index was analyzed by bivariate correlation analysis. Receiver operating characteristic curve(ROC)was utilized for calculating the predictive value, sensitivity and specificity of LUS score for severe PGD.Results:A negative correlation existed between LUS and oxygenation index in single lung transplant( r=-0.536, P<0.01)and a negative correlation between LUS and oxygenation index ( r=-0.518, P<0.01)in double lung transplant. The area under ROC curve of LUS showed that the predictive value of LUS of severe PGD patients with single lung transplant was 7.0 with a sensitivity of 86.7% and a specificity of 72.1%, the predicted value of LUS of severe PGD in double lung transplant was 13.0 with a sensitivity of 83.3% and a specificity of 60.0%. Conclusions:Lung ultrasound is simple and easy to operate and LUS can evaluate the severity of PGD patients after lung transplantation. It has a high potency of guiding clinical diagnosis and treatment.