Objective To construct mycobacterial membrane-anchored expression vector and to analyze expression level and sub-cellualr localization of exogenous target protein. Methods Based on the mycobacterial intracellular expression vector pMFA42 which contained a strong promoter of pfurAma mutant, the signal sequence of Mycobacterium tuberculosis(Mtb) 19×103 lipoprotein (19SS) was synthesized and was then cloned into the downstream of pfurAma mutant to generate the mycobacterial membrane-anchored expression vector pMFA42M. The coding gene of enhanced green fluorescent protein(EGFP) was amplified by PCR, and then sub-cloned into these two vectors described above to construct recombinant EGFP fused and membrane-anchored strains, respectively. The coding genes of Mtb immuno-dominant antigens Ag85A and its chimera Ag856A2 were then sub-cloned intothe membrane-anchored construct pMFA42MG to produce recombinant Mtb antigen EGFP fused-expression strains. After that, expression levels and sub-cellualr localization of exogenous target protein were further analyzed by Western blot and flow cytometry sorting(FCS), and the fluorescence intensities of recombinant EGFP- expressed strains were observed in vitro directly and after transfection of murine macrophage cell line RAW264.7. Results The novel mycobacterial membrane-anchored expression vector was constructed successfully by introduction of signal sequence of Mtb 19×103 lipoprotein. Using of EGFP as model antigen, exogenous target protein was demonstrated to be expressed with high level and could be anchored into cell membrane of recombinant mycobaterial strains. Conclusion A novel mycobacterial membrane-anchored expression vector was constructed successfully to research recombinant BCG and functions of mycobacterial membrane proteins, and the constructed EGFP-expressed recombinant strains could also be used to research cytophagy in cell model and mycobacterial colony and translocation in animal immunization as model indicator bacteria.

Objective To develop a novel padlock probe and rolling circle amplification(RCA) to detect 5 common cell culture contaminant Mollicute( Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycomplasma orale, and Acholeplasma laidlawii ). Methods "Universal" primers ( SPS1, SPA2 and SPS2, SPA1) were used to amplify the Mollicute 16S-23S rRNA intergenic spacer region.Amplicon was ligate Mollicute specific padlock probe. Probe amplified and monitored using a Corbett RotorGeneTM 6000 machine. Results Five reference Mollicute strains were correctly detected by RCA method.There was no cross-reaction. RCA method can sensitive detect 10 copies templates and show strong positive signal. Sixty-two cell culture specimens were detected. Thirty-seven samples were contained single specie Mollicute and 14 samples contained two Mollicutes. Eleven samples did not contained Mollicute. RCA detection results were concordant with previously species-specific PCR. Conclusion RCA can rapid, sensitive and specific detect contamination Mollicute.

AIM: To explore the feasibility of bone tissue regeneration guidance with the frozen heterogeneous periosteum. METHODS: The experiment was conducted in the animal laboratory of Beijing Shijitan Hospital from March to December 2002. ①Preparation of the frozen heterogeneous periosteum: The fresh pigs' cranial or rib bone periosteum was taken and washed with saline solution repeatedly, then defatted by immersion in 0.75 volume fraction alcohol, followed by frozen in the liquor of nitrogen for 10 minutes repeatedly for 3 times. After irradiated under ?-ray up to the amount of 3 kGy, the created membrane was placed in refrigerator for using. ②Surgical procedure: Two artificial mandibular bone defects were performed in 30 adult Japanese white big ear rabbits. One defect was covered with the created membrane as the experimental group, while another as the control group. ③Index observation: The rabbits were sacrificed after 2, 4, 8, 12, 16 weeks postoperatively for macroscopic, radiographic and histopathological observation. RESULTS: All 30 rabbits were involved in the result analysis. ①Macroscopic examination of the two groups: At the eighth week, guiding membrane was observed with irregular border. The experiment group was flat and stiff, while the control group was restored but with soft bone; at the twelfth week, the guiding membrane and surrounding border were unclear, but still could be observed. The newly bone of the experiment group was flat and stiff, while the control group was soft with hollow. ②Histopathological examination: At the eighth week, the new-formed bone in the margin of defect area of the experimental group grew intensively and the inflammatory cells in grafted membrane disappeared; intramembrane fiber was found oedema and broken; at the twelfth week, the trabecular bone of the experimental group was thick and arranged regularly. However, in the control group, the newly formed bone was little in the defect, and the trabecular bone was thinner compared with the experimental group. No evidence of inflammatory reactions was apparent, meanwhile the membrane degraded gradually. ③Radiograph examination: The bone density of the experimental group was greater than the control group at 4 and 8 weeks, and had no significant difference as to the vicinity bone at 12 and 16 weeks; on the other hand, the density of the control group was still lower. CONCLUSION: The frozen heterogeneous periosteum is a useful guiding membrane material for tissue regeneration, because it has no obvious rejection action in body, and could maintain its shape for 8-12 weeks, prevent the fiber tissue from growing into the wound surface, separate different cells and guide tissue regeneration.

Objective To express and purify mouse interleukin 17A(mIL-17A) in E. coli and to analyze its ability of stimulating macrophage inflammatory factors expression. Methods The coding gene of mouse mIL-17A mature protein was amplified from mouse spleen cells by RT-PCR. PCR product was cloned into the prokaryotic expressing vector pET28a, and the resulting recombinant plasmid pET28a/mIL-17a was then transformed into the host E. coli strain BL21(DE3) for expression. The mIL-17A protein was identified by SDS-PAGE and Western blot. The recombinant protein was purified by the Ni-NTA affinity chromatography, and was further tested on the stimulation of cytokine and chemokine of RAW264.7 cells by ELISA and real-time quantity PCR in vitro. Results The mIL-17A with bioactivity was over-expressed and purified successfully, and the results of real-time PCR and ELISA showed that recombinant mIL-17A stimulated macrophage mRNA upregulation of IL-6, defensin β2 and Cxcl3 and secretion of defensin β2, Ccl3, Cxcl3,IFN-γ, IL-6 and IL-4. Interestingly, these effects could be blocked by the addition of anti-IL-17A neutralizing antibody partly. After treatment with mIL-17, 74. 87-fold of defensin β2 mRNA expression was increased comparing with that of untreated cells( P ＜0.01 ), while blocking with anti-IL-17A antibody the increase was only 5.4-fold(P ＜ 0.01 ). Conclusion The recombinant mIL-17A has a strong stimulation on secretion of cytokine and chemokine of macrophage, that maybe result to the enhancement of anti-infection ability of macrophage.

Objective To develop mycobacterial inducible expression vectors which permit to overexpress Mycobacterium tuberculosis (Mtb) immunodominant antigen, and to analyze its immunogenicity after purification by affinity chromatography. Methods The regulatory region of M. smegmatis (Ms) acet-amidase(pACE) was obtained by PCR amplification, and was used as promoter to construct the mycobacteri-al inducible expression vectors, pMF series. The coding gene of Mtb chimeric antigen Ag856A2 which is a recombinant Ag85A with 2 copies of ESAT-6 inserted in its Acc Ⅰ site and showed excellent immunogenicity in the animal experiments we described previously, was cloned into the pMF vector series, and was induced to express by the addition of acetamide. The recombinant protein expressed in the Ms was purified by the Ni~(2+)-NTA affinity chromatography, the resulted homologous recombinant antigen was added into the spleen cells separated from BCG vaccinated mice, and the immunogenicity was analyzed by the IFN-γ ELISPOT as-say. Results The mycobacterial inducible expression vectors, pMF series was constructed successfully, target antigen could be. induced to express in the Ms by the addition of 0.02% acetamide, and could be puri-fied by the Ni~(2+) -NTA affinity chromatography due to the addition of 6×His tag in the vector pMF406. Fur-thermore, the mycobactefial homologous antigen could induce more IFN-γ secretion than the heterogonous one. Conclusion The mycobacterial inducible expression system based on the regulatory region of Ms acet-amidase as promoter could permit the Mtb target antigen of interest overexpression and purification, and the immunogenicity of the homologous antigen from Ms is better than that of be expressed from E. coli, which may be more potential for immunological detection of tuberculosis.

Objective To develop a multiple PCR-based reverse line blot hydrization assay (mPCR-RLB)to simutaneously detect several STD pathogens:Neisserria gonorrhoeae,Chlamydia trachomatis,Ureaplasma urealyticum,U.parvum,Mycoplasma genitalium,M,hominis and Trichomonas vaginalis.Methods Seven pairs of biotin-labelled primer were designed and synthesized to target the 16S rRNA-23S rRNA intergenic spacer regions of Neisserria gonorrhoeae,Chlamydia trachomatis,Mycoplasma,and repetitive DNA sequence of Trichomonas to identify and subtype thesc pathogens.DNA was extracted from the referrence strains of seven pathogens and used as templates.mPCR was performed to simutaneously amplify the target regions of these pathogens.Then,the biotin-labelled amplicons were hybridized with membrane-bound specific oligonucleotide probes followed by the detection of bound amplicons with chemiluminescence assay.Serially diluted plasmids containing the target genes of pathogens were amplified with this method to detect its sensitivity.Two-hundred and eleven specimens,including 104 male urethral swabs and 107 female cervical swabs,were collected from the STD clinic of Wuhan First Hospital;mPCR-RLB and single-primer PCR were performed.For specimens with inconsistent results,nested PCR was performed to confirm the results.Results The assay sensitively and specifically identified referrence strains of the tested pathogens.The detection limit of mPCR-RLB was 100 copies for all the pathogens.Of the 211 clinical specimens,2.8%(6/21)were negative for single-primer PCR,but positive for mPCR-RLB,and nested-PCR results were consistent with those of mPCR-RLB.Conclusion mPCR-RLB is a sensitive,specific and rapid method for the detection of STD pathogens from clinical specimens.

-β2 was positively correlated with OPG (r=0. 432,P<0. 01). Conclusions The reference ranges of serum TGF-β1 and TGF-β2 in healthy adult females are established. Both TGF-β1 and TGF-β2 of them are correlated with OPG and ieptin.

OBJECTIVETo study the anti-cataract effect of gigantol combined with syringic acid and their action mechanism.

METHODH202-induced lens oxidative injury in vitro rat model was establish to observe the impact of gigantol combined with syringic acid on lens transparency under a dissecting microscope. D-galactose-induced cataract rat model was established to observe the impact of gigantol combined with syringic acid on lens transparency under a slit-lamp. UV spectrophotometry was adopted to detect the inhibitory activity of gigantol combined with syringic acid against AR. Molecular docking method was used to detect binding sites, binding types and pharmacophores of gigantol combined with syringic acid in prohibiting aldose reductase.

RESULTBoth in vitro and in vivo experiments showed a good anti-sugar cataract activity in the combination of gigantol and syringic acid and a better collaborative effect than single component-gigantol and syringic acid and positive control drug Catalin. Molecular docking and dynamic simulation showed their collaborative AR-inhibiting amino acid residue was Asn160 and the major acting force was Van der Waals' force, which formed common pharmacophores.

CONCLUSIONGigantol combined with syringic acid shows good anti-cataract, their action mechanism is reflected in their good collaborative inhibitory effect on AR.

Aldehyde Reductase ; antagonists & inhibitors ; Animals ; Bibenzyls ; Cataract ; drug therapy ; enzymology ; Drug Synergism ; Female ; Gallic Acid ; analogs & derivatives ; pharmacology ; Guaiacol ; analogs & derivatives ; pharmacology ; Humans ; In Vitro Techniques ; Lens, Crystalline ; drug effects ; enzymology ; Male ; Rats ; Rats, Wistar

Objective To evaluate the effects of simulation training based on the concept of experiential education on im-proving first aid skills of medical staff in nursing homes.Methods The assessment data of 120 medical staff participating in first aid simulation training in our nursing home from January 2020 to June 2023 were retrospectively analyzed.Using a self-controlled design,the changes in skills operation,reaction time,team cooperation,etc.were statistically analyzed.Results All indicators were significantly improved after training.The skill operation score increased by 12.4 points(P＜0.05),the average reaction time decreased by 5.2 seconds(P＜0.05),and the team cooperation score increased by 13.8 points(P＜0.05)compared with before training.Conclusion Simulation training based on the experiential education model can effectively improve the first aid skills and cooperation ability of medical staff,which is an important means to continuously improve the emergency response capac-ity in nursing homes.

Objective To compare the difference of cutpoint and clinical significance of HbA1C for the diagnosis of abnormal glucose metabolism in two population groups with different ages.Methods According to oral glucose tolerance test(OGTT),the cutpoint and clinical significance of HbA1C for the diagnosis of type 2 diabetes and impaired glucose regulation(IGR)were investigated in the two population groups.Results The mean HbA1C of 1 064 young subjects in an academy and 1 671 aged subjects in a community were 5.31% ±0.41% and 5.79% ±0.71%,respectively.The cutpoints of HbA1C for diagnosis of diabetes were 5.7%(specificity 86.7%,sensitivity 66.7%)and 5.9%(specificity 73.8%,sensitivity 80.1%)in the two population groups,and 5.6% for diagnosis of IGR (specificity 82.8%,sensitivity 55.8%)and 5.7%(specificity 60.9%,sensitivity 64.3%),respectively.87.8%,78.7%,and 38.5% were diagnosed diabetes by current OGTT criteria at HbA1C levels of ≥5.7%,≥5.9%,and≥6.5%,IGR being 61.6%,39.6%,and 4.1%,and normal glucose tolerance being 24.4%,10.0%,and 0.4%.Conclusion The cutpoints of HbA1C for diagnosis of diabetes and IGR are different in populations with different ages and HbA1C levels.As one of diagnostic criteria for diabetes,HbA1C 6.5% with relatively higher specificity and lower sensitivity must be combined with fasting blood glucose,random blood glucose,and OGTT.