Objective To compare the effects of different signal peptides on the secretion and expression of SARS-CoV-2S1,receptor binding domain(RBD) and RBD dimer proteins in Expisf9 insect cells.Methods The gene sequences of three proteins,SARS-CoV-2 S1(M1-E661),RBD(R319-P545) and RBD dimer(R319-K537 tandem),were selected and divided into 25 groups according to the different N-terminal signal peptide sequences(Endo,honeybee melittin(HBM),GP64,GP67,chitinase(Chi) and HIV-ENV) and C-terminal label sequences.25 recombinant baculoviruses were constructed by Bac-to-Bac system,and 25 groups of tertiary strain banks were prepared.B2 and C4 viruses were inoculated to logarithmic prestage cells(2.8 × 10~6 cells/mL) and logarithmic metaphase cells(1.2 × 10~7 cells/mL),respectively.The viruses of each group were cultured to 100 mL(500 mL shaker) for protein expression,and samples were taken for SDSPAGE electrophoresis,Western-blot and ELISA detection.Two groups with higher expression levels of S1,RBD and RBD dimer proteins were selected for repeated verification.Results When B2 and C4 were inoculated to high cell density,the secretion expression level showed no increase,while there were significant difference between 4 and 5 d after inoculation.The expression level of A7(Endo-S1-tag) was significantly lower than that of A9(HIV-ENV-S1-tag),the expression level of A4(Gp67-S1-tag) was the highest,and the secreted expression level of A1(Endo-Endo-Sl-tag) was significantly lower than that of A7(Endo-S1-tag).The secretion and expression of B6(HIV-ENV-RBD-tag) was signifi-cantly higher than that of B4(Gp67-RBD-tag) and other signal peptide groups,and C4(Gp67-RBD-dimer-tag) expression was significantly higher than that of C3(Gp64-RBD-dimer-tag).Two groups with high expression of each protein were selected separately for repeated verification(A4,A9;B4,B6;C3,C4) and the results showed that A4,B6 and C4 had the highest secretion expression levels.Conclusion The signal peptide for the highest secretion expression of S1 and RBD dimer proteins is the same,which is GP67 signal peptide,while the most suitable signal peptide for RBD protein is HIV-ENV,indicating that the N-terminal sequence can affect protein secretion,signal peptide sequence is universal to a certain extent,but is also related to the target protein sequence to be expressed.

Objective To compare the effects of different signal peptides on the secretion and expression of SARS-CoV-2S1,receptor binding domain(RBD) and RBD dimer proteins in Expisf9 insect cells.Methods The gene sequences of three proteins,SARS-CoV-2 S1(M1-E661),RBD(R319-P545) and RBD dimer(R319-K537 tandem),were selected and divided into 25 groups according to the different N-terminal signal peptide sequences(Endo,honeybee melittin(HBM),GP64,GP67,chitinase(Chi) and HIV-ENV) and C-terminal label sequences.25 recombinant baculoviruses were constructed by Bac-to-Bac system,and 25 groups of tertiary strain banks were prepared.B2 and C4 viruses were inoculated to logarithmic prestage cells(2.8 × 10~6 cells/mL) and logarithmic metaphase cells(1.2 × 10~7 cells/mL),respectively.The viruses of each group were cultured to 100 mL(500 mL shaker) for protein expression,and samples were taken for SDSPAGE electrophoresis,Western-blot and ELISA detection.Two groups with higher expression levels of S1,RBD and RBD dimer proteins were selected for repeated verification.Results When B2 and C4 were inoculated to high cell density,the secretion expression level showed no increase,while there were significant difference between 4 and 5 d after inoculation.The expression level of A7(Endo-S1-tag) was significantly lower than that of A9(HIV-ENV-S1-tag),the expression level of A4(Gp67-S1-tag) was the highest,and the secreted expression level of A1(Endo-Endo-Sl-tag) was significantly lower than that of A7(Endo-S1-tag).The secretion and expression of B6(HIV-ENV-RBD-tag) was signifi-cantly higher than that of B4(Gp67-RBD-tag) and other signal peptide groups,and C4(Gp67-RBD-dimer-tag) expression was significantly higher than that of C3(Gp64-RBD-dimer-tag).Two groups with high expression of each protein were selected separately for repeated verification(A4,A9;B4,B6;C3,C4) and the results showed that A4,B6 and C4 had the highest secretion expression levels.Conclusion The signal peptide for the highest secretion expression of S1 and RBD dimer proteins is the same,which is GP67 signal peptide,while the most suitable signal peptide for RBD protein is HIV-ENV,indicating that the N-terminal sequence can affect protein secretion,signal peptide sequence is universal to a certain extent,but is also related to the target protein sequence to be expressed.

There are some unsafe and inconvenient problems in moving Ⅴb-type field tents because the overall weight amounts to 310Kg,and the medical boxes are lack of sign cards and block slots.Improving methods and suggestions for these problems are put forword.

Objective To investigate the effects of propefol on β-amyloid protein(β-AP)-induced injury to cultured rat cortical neurons.Methods Eighteen days pregnant SD rats were anesthetized with ether.The fetal rats were obtained under sterile condition and decapitated. Cortices were then dissected under dissecting microscope.Cortical neurons were isolated according to the method described by Velly LJ et al and cultured for 7 days.There were 5×104 neurons in each well (200 μl).The experiment included 2 parts.In part T 15 wells of neurons were randomly divided into 5 groups(n=3 each ) : group I control(C);group II β-AP 25 μmol/L; group III and IV 2 propofol pretreatment groups (PP1,PP2) and greup V propofol treatment (PT).In group PPt propofol 50 μmol/L was added to the culture medium 24 h before the addition of β-AP 25 μmol/L and the neurons were incubated for another 24 h.In group PP2 propofol 50 μmol/L and μ-AP 25 μmol/L were added to the culture medium simultaneously and the neurons were then incubated for 24 h.In group PT propefol 50 μmol/L was added to the culture medium at 6 h after the addition of β-AP 25 μmol/L and the neurons were incubated for another 18 h.In part Ⅱ 18 wells of neurons were randomly divided into 6 groups(n=3 each):group I control (C) ; group IIβ-AP 25 μmol/L; group III intralipid; group IV,V,and VI 3 prepofol treatment groups (P1,P10,P50).In intralipid group equal volume of 10% intralipid was added to the culture medium at 6 h after β-AP 25 μmol/L and the neurons were then incubated for another 18 h.In group IV- VI propofol 1,10 and 50 μmol/L were added at 6 h alter β-AP 25 μmol/L respectively and the neurons were incubated for another 18 h.The amount of lactic dehydrogenase (LDH) released was measured.Neuronal viability was assessed by MTT assay.The neuronal apoptosis was detected using Hoechst33342 staining and TUNEL technique,and the.apoptosis rate was calculated.Results In part Ⅰ there was no significant difference in the amount of LDH released between group Ⅱ(β-AP) and the 2 propofol pretreatment groups(Ⅲ,Ⅳ).The amount of LDH released was significantly lower in group Ⅴ (propofol treatment) than in group β-AP(Ⅱ).In part Ⅱ the amount of LDH released was significantly lower,neuronal viability higher and the apoptosis rate was lower in group P50 than in group Ⅱ(β-AP).Conclusion Propofol 50 μmol/L given after β-AP can attenuate β-AP induced injury to cultured rat cortical neurons while prophylactic administration of propofol can't.

Objective To investigate the clinical character and treatment method of the coexistence of cervical and lumbar spinal stenosis.Method Twenty-seven cases with coexistence of cervical and lum bar spinal stenosis,9 cases with single cervical operation,7 cases with single lumbar operation,11 combined cervical and lumbar operation.Results Twenty-two cases were followed-up,average 49 months,all eases were recorded with JOA score.JOA score was(8.6±0.2)scores preoperafion,(11.7±0.3)scores 2 weeks postoperation,(13.1±0.2)scores 3 months postoperation,(13.5±0.2)scores 6 months postoperation,(13.9±0.3)scores 12 months postoperation.Relief rate was 79.2%,satisfactory rate was 93.8%.Conclusions The patient with multi-segment spinal stenosis should be treated individually,to certain responsible focus.If no responsible focus,it is better to treat with cervical spinal operation first.

Objective To evaluate the effects of Burch-Schneider(BS) acetabular reinforcement ring applied in primary or revision total hip arthroplasties in 9 patients with combined or cavitary acetabular bone defects. Methods A retrospective follow up that averaged 3.8 years was performed with a special scoring system comprising the patients symptoms, signs and function. Results The excellent and good function were achieved in 89% of all cases. Conclusion 1)BS reinforcement ring could be effectively and reliably applied in primary or revision total hip arthroplasty or in patients with severe acetabular bone defects. 2)BS reinforcement ring shows the biological fixation feature, so as to provide good foundation for long-term satisfactory function of hip. 3)The lower ear of BS ring need not to be fixed to ischium with screws as usual, but be inserted in obturator foramen above transverse ligament, thus imparting some elastic resilience to it. 4)The upper ear should be folded through such a curve that its curvature was in compliance with that of ilium, which would be better performed in one session. 5)The appropriate amount of 0.8 cm?0.8 cm?0.8 cm morcellized allografting bone was used to snugly fill the space, which would be conducive to desirable incorporation between prosthesis and bone, and the authors propose that the patients should be allowed weight-bearing earlier.

Objective To improve the management and utilization of hospital sterilizing devices. Method Strict and operable managerial system was established. Result Hospital sterilizing devices behaved well. Conclusion Just like the purchase, utilization and maintenance of large-scale equipments, the ones of hospital sterilizing devices also should be integrated to optimize their performances.

According to the recognition of medical engineering department after the army reform,this paper puts forward some suggestions on the transform and development of medical engineering function.

Objective To compare the clinical effects of small splint fixation after closed manipulative reduction with plaster external fixation in the treatment of elderly type C Colles fractures.Methods Totally 96 elderly patients with type C Colles fractures were randomly divided into two groups, 48 patients in each group. Both groups were treated with closed manipulative reduction. The fractures were externally fixed with splint in the treatment group, and those were externally fixed with pipe plaster in the control group. The figure of radius length, palmar tilt and ulnar deviation were detected respectively in pre-reduction, the same day with reduction, and the day when external fixation was removed (6th week), in order to evaluated the drop of anatomical position. The therapeutic effects were evaluated according to Gartland-Werley wrist score when external fixation was taken off after 6 months.Results In both groups, figures of radius length, palmar tilt and ulnar deviation evidently increased in the day after reduction and the day of external fixation removal, compared with pre-reduction (P<0.05). Compared with the day after reduction, the radius length, palmar tilt and ulnar deviation of the day of external fixation removal all dropped in both groups (P<0.05). But the drop degree in control group was significantly higher than treatment group (P<0.05). For the joint function, 9 cases for excellent, 26 for good, 6 for fair, and 4 for bad in the therapy group. The excellent and good rate was 77.8% (35/45). In the control group, 4 cases for excellent, 22 for good, 10 for fair and 9 for bad. The excellent and good rate was 57.8% (26/45). The curative effect of treatment group was remarkably superior to control group (P<0.05).Conclusion Closed manipulative reduction combined with splint external fixation is more effective than pipe plaster in treatment of elderly type C Colles fractures.

[Objective] To investigate the characteristics of PD-1/PD-L1 expressed on T cell and bladder cancer cell and clinical significance.[Methods] 64 patients with primary bladder cancer were into experiment group and 10 normal people were into control group.Peripheral bloods were used to test the PD-1 expressed on CD8+ T lymphocytes by flow cytometry.Immunohistochemistry staining was used to detect the PD-L1 expression in tumor and normal specimen.[Results] PD-1 expressed on CD8+T lymphocytes was (2.25 ± 0.60)％ in experiment group and (0.68 ± 0.17)％ in control group,respectively (P ＜ 0.001).And the PD-1 expression on T cell in invasive bladder cancer patient was significant higher than superficial bladder cancer patients [(3.04 ± 0.46)％ vs (0.68± 0.17)％,P ＜ 0.001].The expression of PD-L1 in experiment group was higher than control group,(26/64 vs 0/15,P ＜ 0.001).But there was no different between invasive and superficial bladder cancer patients,(41.3％ vs 38.8％,P ＞ 0.01).[Conclusions] Expression of negative stimulatory molecule PD-1 in CD8+T lymphocytes of peripheral blood is significantly correlated with bladder cancer advanced.Bladder cancer cell was strongly expressed PD-L1,and this expression is not related to cancer advanced.