Objective To construct a prokaryotic expression system of human ferritin L which could be overexpressed in E.coli, and then prepare quality control materials of rFL and evaluate the homogeneity, stability, precision and application value of the rFL.Methods RT-PCR was used to clone ferritin L gene with total RNA from peripheral blood.Ferritin L gene was inserted into plasmid pGM-T to construct subclone plasmid pGM-T/ferritin L, which was identified by sequencing.The recombinant plasmid pET-30a/ferritin L was transformed into E.coli BL21 (DE3) and efficiently expressed under IPTG induction.rFL was identified by SDS-PAGE, and its antigenicity was examined by WB.The concentration of rFL was measured by ACCESS immunoassay system.Fifteen control samples were randomly selected using random number table.The homogeneity and stability of rFL were evaluated by one-way analysis of variance (ANOVA) and a linear regression model, respectively.The uncertainties of homogeneity, stability and the precision were evaluated.Results A prokaryotic expression system of ferritin L was successfully constructed.Sequence analysis showed that the ferritin L gene inserted was identical with the one from GenBank(NM_000146.3).The PCR products were 527 bp long, in complete agreement with length of ferritin L gene.The molecular weight of rFL highly expressed in E.coli induced by IPTG was 19 000 Da.The WB analysis indicated that this rFL protein had good antigenicity.The concentration of rFL(1 000 times dilution) measured by ACCESS immunoassay system was (1115.84±38.38) ng/ml.Homogeneity evaluation of low-concentration QC sample and high-concentration QC sample of rFL showed that there were no statistical significances in the within-groups and between-groups (for high-concentration F=2.336, P>0.05 and for low-concentration F=0.730, P>0.05).A linear regression based on the stability test indicated that there was no statistically significant trend of instability in five and a half months (F=1.755, P>0.05). Precision analysis showed the within-run CV, the between-run CV, the between-day CV and the total CV of high-concentration QC sample were 2.06%, 2.12%, 0% and 2.96% respectively; the within-run CV, the between-run CV, the between-day CV and the total CV of low-concentration QC sample 2.03%, 2.08%, 0% and 2.90%, respectively.Conclusion This rFL for quality control materials with independent intellectual property rights could meet the clinical requirement of homogeneity, stability and precision, and could provide raw materials for preparation of mixed ferritin for quality control.

Objective To establish the training objective systems for undergraduates in mili-tary clinical medicine in order to promote the educational reform of military medicine, and to improve the quality of talents training in military medical universities. Methods Literature analysis was made to determine the initial military clinical medical undergraduate training objectives, and a questionnaire was given to experts, on-the-job military doctors, medical graduates to know the real requirements of military doctors' position. A total of 455 valid questionnaires were collected. Then the questionnaire results were discussed and summarized by the method of expert discussion. Finally,. Delphi method was used to determine the ultimate training objective system. Results The ultimate training objective system of military undergraduate clinical medicine contains two aspects, eight fields and sixty-one standards objective systems. Conclusion Organically fusing military medical knowledge, ability and professional quality requirement into two levels and eight modules and clearly understanding the standards and requirements of each module are significantly systematic, advanced and operable, which has important reference value and guiding significance to both the military medical colleges and uni-versities and the local universities.

Objective To study the in vitro culture condition for melanoblasts from human foreskin tissue. Methods The skin tissue taken from foreskin of children was treated with 0.5% dispase Ⅱ to separate epidermis from dermis, then with trypsin to obtain single cell suspension, which was cultured in modified medium for melanoblasts, i.e., MCDB254 medium supplied with several cell growth factors. Finally, melanoblasts were obtained based on the difference of adhesion speed. The morphology and proliferation of cultured melanoblasts were observed under a light microscope. DOPA staining, immunostaining with anti- S-100 and -tyrosinase related protein 2 (TRP2) antibodies, and transmission electron microscopy were per- formed to identify the cultured melanoblasts. Results The cultured human melanocytes displayed a match-like shape, scattered arrangement, syrmnetric double poles, slim cell body, highly refractive nuclei; meanwhile, the melanoblasts exhibited plentiful cytoplasm, large volume, bipolar or irregular shape and clonal growth. Additionally, the melanocytes were positive for TRP2, S-100 and Dopa staining, while the melanoblasts were positive only for TRP2. Electron microscopy revealed the presence of mature melanin granules (stage Ⅲ-Ⅳ ) in melanocytes but immature melanin granules (stage Ⅰ ) in melanoblasts. Conclu- sion Stable pure culture of melanoblasts has been realized with the reformed medium, which may lay a foundation for the investigation into the mechanism of epidermal pigmentation.

Objective:To study the effect of Qingyi Lidan Granule on the serum levels of high mobility group box 1 (HMGB 1),heat shock protein 70 (HSP70),heat shock protein 27 (HSP27) and interleukin-8 (IL-8) of patients with severe acute pancreatitis.Methods:From August 2015 to July 2016,84 patients with severe acute pancreatitis in our hospital were selected and randomly divided into the observation group and the control group according to random number,42 cases in each group.The control group was given routine treatment,and the observation group was treated by Qingyi Lidan Granule on the basis of control group.The recovery of blood amylase to normal time,white blood cell recovery to normal time,recovery of gastrointestinal function to normal time and relieving time of abdominal pain,serum levels of HMGB1,HSP70,HSP72 and IL-8 in both groups were observed and compared before and after treatment.Results:After treatment,the total clinical effective rate of observation group was significantly higher than that of the control group[92.86％ (39/42) vs 71.43％ (30/42)] (P＜0.05).The recovery of blood amylase to normal time,white blood cell recovery to normal time,recovery of gastrointestinal function to normal time and relieving time of abdominal pain in the observation group were significantly shorter than those of the control group (P ＜0.05).Before treatment,no significant difference was found in the serum levels of HMGB 1,HSP70,HSP72,IL-8 between groups (P＞0.05).After treatment,the serum levels ofHMGB1,HSP70,HSP72 and IL-8 in both groups were significantly lower than those before treatment (P＜0.05).The levels ofHMGB1,HSP70,HSP72 and IL-8 in the observation group were lower than those in the control group (P＜0.05).Conclusion:Qingyi Lidan Granule could effectively reduce the levels of serum HMGB 1,H SP70,HSP27 and IL-8 and enhance the clinical curative effect of patients with severe acute pancreatitis.

BACKGROUND: Most reported biomechanical studies on sacroiliac joint injury and fixation use cadavers or artificial bone models to simulate the sacroiliac joint injury.OBJECTIVE: To analyze the vertical stability of anterior plate fixation for sacroiliac joint dislocation using three-dimensional finite element method. METHODS: The anterior plate fixation model of unilateral sacroiliac joint dislocation was constructed on the basis of the three-dimensional finite element model of a complete pelvis. An axial load of 500 N was applied on the model; the cloud pictures of stress, strain and displacement were obtained after calculation and compared with that of the complete pelvis under the same conditions.RESULTS AND CONCLUSION: Stress concentration occurred at the internal fixation system; the maximum stress was found at the screws near the injured sacroiliac joint, far greater than the maximum stress of the complete pelvis under the same condition. The maximum strain was found in the healthy sacroiliac joint; the fixed sacroiliac joint had no strain. The maximum displacement was found in the injured sacroiliac joint; it was about twice longer than the complete pelvis. These findings indicate that the vertical stability of pelvis is poor using anterior plate internal fixation treatment for sacroiliac joint dislocation; and stress concentration occurs at the screws and plates.

Objective To establish a chromogenic assay for blood-plasma collagenase Ⅳ in order to evaluation the reference range of collagenase Ⅳ in the plasma of healthy individuals.Methods The assay was based on measurement of terminal amino group with succinylated gelatin as substrates and TNBS as chromogenic reagent.The optical density of each reaction was determined at 405 nm using a Sunrise microplate reader.Chromatographic and detection conditions were optimized and performance of the methed was evaluated by recovery experiments and precision experiments.It was compared with ELISA.The levels of collagenase Ⅳ in 112 health persons'plasma were determined and the data were analyzed by SPSS statistical software.Results The whole determing time was within 1.5 h,the linear range of this method was 1.5-10.0 mg/L,and the minimum detection limit was 0.965 mg/L.They were well correlated with ELISA results (R~2=0.999 7,P＜0.01).The within-run CV was less than 3.16%and between-run CV was less than 9.81%.The 95%confidence interval of collagenase Ⅳ in healthy plasma was 33.38-49.80 mg/L.Conclusion This chromogenic assay for blood-plasma collagenase Ⅳ can be used for measurement of collagenase Ⅳ in blood-plasma and the reference range of collagenase Ⅳ in healthy plasma was established.

Abstract: Objective To observe the phenotypic characteristics of 3 wild-type plague phages under different experimental environments, providing scientific evidence for the identification of phage biological characteristics and the study of their interaction with host bacteria in the future. Methods The sensitivity of 3 wild-type plague phages were detected by using liquid culture method, emisolid medium method and micro-liquid culture method based on OmniLog TM microbial identification system. Results The growth result based on LB liquid medium showed that the growth of plague phage 476 for 20-24 hours at both 28 ℃ and 37 ℃was better than that of plague phages 087 and 072204 at 37 ℃, and the growth of plague phages 087 was better than that of plague phages 072204 at 37 ℃. With the attenuated plague bacterium EV76 as the host bacterium, phage 476 was able to form visible plaque on double-layer agar medium for 20-20 hours at both 28 ℃ and 37 ℃, phages 087 and 072204 were only able to form opaque plaque on double-layer agar medium for 20-24 hours at 37 ℃. The growth results based on OmniLogTM system showed that when plague phage was lysed in EV76 strain at 33 ℃, the first row appeared as a straight line with a peak of no more than 100 in the 96-well microplate curve chart. As the phage quantity decreased, the dilution plate appeared with growth curve similar to EV76 strain in turn, and the color of tetrazolium dyes in the experimental wells gradually deepened as the phage number decreased and the host bacteria number increased. Therefore, it indicates that phage 476 was sensitively at both 28 ℃ and 37 ℃, while phage 087 and 072204 were temperature-dependent only at 37 ℃ to attenuated plague bacterium EV76. Conclusions The lysing ability of 3 wild-type plague phages are temperature-dependent, and the growth results are consistent under the three experimental conditions.

Caliciviridae Infections ; epidemiology ; Child, Hospitalized ; Child, Preschool ; China ; epidemiology ; Female ; Gastroenteritis ; epidemiology ; virology ; Humans ; Infant ; Male ; Sapovirus ; isolation & purification

Adenoviridae ; classification ; genetics ; pathogenicity ; Adenoviridae Infections ; epidemiology ; prevention & control ; virology ; Age Distribution ; Child, Preschool ; Humans ; Infant ; Infant, Newborn

Objective To observe the surface structures of cardiovascular endothelial cells in situ with atomic force microscope (AFM). Methods Fresh aorta and aortic valve were dissected from 10 healthy male New Zealand white rabbits. Before fixed in 1% formaldehyde, the fresh tissues were washed in the buffer phosphate solution. Under general microscope, the fixed aorta or valve was spread on the double side stick tape which had already been stuck on the glass slide. The intima of aorta or the aorta side of valve was towards upside. Then the specimen was dried under 37 degrees centigrade in an attemperator and was washed with pure water. After dried again, the specimen was loaded on the platform ofNanoScope llla AFM and was scanned in tapping mode with the scanning speed of 0.5 HZ. Results The surface structures of endothelial cell on the fixed and dried tissue could be obsserved clearly in situ with AFM. Aortic endothclial cells were large, branched and arranged sparsely and parallel to the direction of blood flow, whereas endothelial cells on aorta valve surface were small, less branched and arranged intensively and vertical to the direction of blood flow. When the scanning range was dwindled, granular ultra-structures could be observed on the surface of endothelial cells, and, as the scanning range was dwindled further, fissure and convolution could be seen on the surface of granules from aortic endothelial cells. Centre cavity and surrounding swelling volcano-like structure could be seen on the surface of granules from endothelial cells of aortic valve. Conclusions It's feasible to observe the surface ultra-structures of cardiovascular endothelial cells in situ with AFM and morphological information provided by A FM might be of clinical value in future histopathological diagnosis.