BACKGROUND:Exosomes,as a type of extracellular vesicle,have become a key medium for cell-to-cell communication due to their nanoscale size and enrichment of various bioactive substances.The study of exosome secretion regulation not only has important scientific value,but also has broad application prospects in clinical practice,and is of great significance for promoting medical progress and improving human health.OBJECTIVE:To review the biological characteristics,biological functions,biogenesis process and biochemical regulation mechanism of exosomes,and to explore the application prospects of exosomes in disease diagnosis,treatment and vaccine development,so as to provide theoretical basis and reference for basic research and clinical transformation of exosomes.METHODS:The first author searched PubMed and CNKI databases in October 2024 for relevant literature published from January 2010 to October 2024.Key words were"exosomes,biological functions,biogenesis,secretion or release,regulatory mechanisms,application prospects"in Chinese and English.Finally,92 articles were included for analysis.RESULTS AND CONCLUSION:The secretion level of exosomes can be regulated through physical or biochemical means.Exosomes show broad application prospects in the fields of disease diagnosis,treatment,and vaccine development,and may play a key role in the treatment of cardiovascular and cerebrovascular diseases as well as cancer.This review provides valuable information for the clinical translation and application research of exosomes,helping to promote future progress in exosome research and application.

BACKGROUND:Exosomes,as a type of extracellular vesicle,have become a key medium for cell-to-cell communication due to their nanoscale size and enrichment of various bioactive substances.The study of exosome secretion regulation not only has important scientific value,but also has broad application prospects in clinical practice,and is of great significance for promoting medical progress and improving human health.OBJECTIVE:To review the biological characteristics,biological functions,biogenesis process and biochemical regulation mechanism of exosomes,and to explore the application prospects of exosomes in disease diagnosis,treatment and vaccine development,so as to provide theoretical basis and reference for basic research and clinical transformation of exosomes.METHODS:The first author searched PubMed and CNKI databases in October 2024 for relevant literature published from January 2010 to October 2024.Key words were"exosomes,biological functions,biogenesis,secretion or release,regulatory mechanisms,application prospects"in Chinese and English.Finally,92 articles were included for analysis.RESULTS AND CONCLUSION:The secretion level of exosomes can be regulated through physical or biochemical means.Exosomes show broad application prospects in the fields of disease diagnosis,treatment,and vaccine development,and may play a key role in the treatment of cardiovascular and cerebrovascular diseases as well as cancer.This review provides valuable information for the clinical translation and application research of exosomes,helping to promote future progress in exosome research and application.

Objective:To investigate the outcomes of endoscopic balloon dilation laryngoplasty （EBDL） in managing acquired subglottic stenosis in children. Methods:A retrospective analysis of clinical data from patients who underwent endoscopic balloon dilation for secondary subglottic stenosis between January 2017 and January 2024 at Department of Otorhinolaryngology Head and Neck Surgery, Children's Hospital of Fudan University, Shanghai. The study included 10 children （6 males, 4 females） aged between 13 days and 3 years at the time of their first procedure, with an average age of 7 months. Subglottic stenosis was graded according to the Myer-Cotton classification, with two cases classified as grade Ⅱ and eight cases as grade Ⅲ. All patients had a history of tracheal intubation, including seven for rescue purposes and three for operations. Eight cases were complicated by other conditions: two with atrial septal defect, patent ductus arteriosus, and patent foramen ovale; two with patent foramen ovale only; one with atrial septal defect and extreme deafness in the left ear; one with a brain tumor and hydrocephalus; one with a traumatic diaphragmatic hernia and hepatic rupture; and one case complicated by type Ⅰ laryngeal cleft. Prior to surgery, all children required respiratory support-seven needed high-flow oxygen while three required CPAP. Results:All ten cases underwent endoscopic balloon dilation under spontaneous respiration and general anesthesia, totaling fourteen dilations （an average of 1.4 dilations per person） without any complications. Post-surgery air permeability tests showed that eight cases had grade Ⅰ stenosis while two had grade Ⅱ stenosis. The follow-up period ranged from six months to six years （average duration: 46 months）. Following treatment, all patients no longer required respiratory support or experienced significant mobility limitations. Conclusion:Endoscopic balloon dilation under general anesthesia is deemed safe and effective in treating secondary subglottic stenosis. Early diagnosis coupled with prompt intervention can help avoid tracheotomy procedures altogether. Standard tracheoscopy combined with breathability testing represents a crucial approach to assess normal airway diameter and effectively reduce or prevent secondary subglottic stenosis following re-intubation.
Humans ; Laryngostenosis/surgery* ; Male ; Female ; Retrospective Studies ; Laryngoplasty/methods* ; Child, Preschool ; Infant ; Dilatation/methods* ; Laryngoscopy/methods* ; Treatment Outcome ; Endoscopy

To explore the application effect of host restriction factors(HRFs)in the development of antiviral gene drugs,this study based on the"common pathway of viral infection"and"host innate immunity HRFs",the genes of three antiviral HRFs,namely cholesterol-25-hydroxylase(CH25H),interferon-induced transmembrane protein(IFITM3)and interferon-stimulated gene 15(ISG15)were fused and expressed using pCK vectors to construct antiviral gene drugs.The fusion expression gene sequence CH25H-IFITM3-ISG15(C Ⅱ)was constructed by linking the gene cod-ing sequences of these three HRFs through the cleavage peptide coding sequence.Subsequently,the C Ⅱ sequence was amplified by PCR,ligated to the pCK expression vector,and the recombinant plasmid was transformed,identified,and extracted to obtain a candidate biodrug based on the DNA expression system,which was named pCK-CⅡ.Then,the recombinant plasmid was transfected in-to HEK 293T cells,and the expression of three antiviral proteins was successfully detected by Western blot.To clarify the antiviral effect of pCK-C Ⅱ at the cellular level,pCK-C Ⅱ was trans-fected into HEK 293 cells and BHK-21 cells,respectively.Twenty-four hours later,the BHK-21 cells were infected with vesicular stomatitis virus expressing green fluorescent protein(VSV-GFP),12 h later,the cells were observed under a fluorescence microscope and detected by flow cy-tometry;HEK 293 cells were inoculated with H3N2 subtype influenza virus,and the expression of H3N2 subtype influenza virus nuclear proteins was detected after 12 h using Western blot.The re-sults showed that transient transfection of pCK-C Ⅱ plasmid could significantly reduce the fluores-cence level of cells and the expression of nuclear protein of H3N2 subtype influenza virus in infec-ted cells.These results indicated that pCK-C Ⅱ had an inhibitory effect on the infection of VSV-GFP and H3N2 subtypes of influenza viruses after transient transfection of cells.

The synthetic PDCoV N protein gene was optimized and cloned into the pET-30a vector to obtain the pET-30a-N plasmid.Thenthe recombinant plasmid was transformed into three strains of BL21 E.coli using heat-shock to explore protein expression conditions.The expressed proteins was purified using Ni Focurose 6FF(IMAC)and used as antigen to immunize the New Zealand White rabbit to prepare the polyclonal antibody against the PDCoV N protein.The antibody titer was measured by indirect ELISA method.The specificity for the antibody was identified by West-ern blot and indirect immunofluorescence(IFA).The results showed that the pET-30a-N plasmid showed high expression level in BL21 StarTM(DE 3).The optimal expression condition was 37 ℃ 4 h.The purity of the target protein could reach 90.3％after purification.Indirect ELISA showed that the antibody titers was up to 1∶204 800.Western blot and IFA showed that the produced rabbit polyclonal antibody exhibited good specificity.In conclusion,the polyclonal antibody was prepared which specifically recognized the PDCoV N proteins.The results provided some references for the subsequent exploration of PDCoV N protein function and laid a foundation for establishing a diag-nostic method for PDCoV.

This study aims to screen epitope antigens targeting the receptor binding domain(RBD)of porcine epidemic diarrhea virus(PEDV)based on its amino acid sequence(GenBank accession number:AKN45969.1),prepare PEDV RBD polyclonal antibody,and perform their identification.Bioinformatics analysis software was used to predict the potential antigenic epitopes of PEDV RBD and sequence comparison with porcine coronavirus strains was performed,the selected dominant antigen epitopes were then conjugated with keyhole limpet hemocyanin(KLH),to synthesize pep-tides directly and immunize mice to generate specific antibody,Western blot technique and indirect immunofluorescence assay were utilized to identify the specificity of the antibodies,and indirect ELISA method was further applied to determine the antibody potency.Results showed the selected PEDV RBD dominant epitope sequence shared 100％similarity with 18 other PEDV strains,while exhibiting low sequence similarity with 11 TGEV strains(27.8％—29.3％)and 16 PDCoV strains(10.5％—13.4％),indicating good epitope conservation.Western blot showed that the specificity of the prepared peptide antibody specifically recognized the PEDV RED protein overexpressed in Ex-pi293F cells and overexpressed in baculovirus system,and at the same time,the antibody was still able to detect the PEDV S protein expressed in PEDV-infected Vero cells at a 1∶2 000 dilution,while it did not react with TGEV-and PDCoV-infected ST cells,indicating that the good specificity of the peptide antibody.ELISA revealed that the potency of specific antibodies in mouse serum could reach up to 1∶25 600.The above results indicate that bioinformatics techniques were suc-cessfully utilized to predict antigenic epitopes of PEDV RBD protein,and specific PEDV RBD pep-tide antibodies were prepared.

The synthetic PDCoV N protein gene was optimized and cloned into the pET-30a vector to obtain the pET-30a-N plasmid.Thenthe recombinant plasmid was transformed into three strains of BL21 E.coli using heat-shock to explore protein expression conditions.The expressed proteins was purified using Ni Focurose 6FF(IMAC)and used as antigen to immunize the New Zealand White rabbit to prepare the polyclonal antibody against the PDCoV N protein.The antibody titer was measured by indirect ELISA method.The specificity for the antibody was identified by West-ern blot and indirect immunofluorescence(IFA).The results showed that the pET-30a-N plasmid showed high expression level in BL21 StarTM(DE 3).The optimal expression condition was 37 ℃ 4 h.The purity of the target protein could reach 90.3％after purification.Indirect ELISA showed that the antibody titers was up to 1∶204 800.Western blot and IFA showed that the produced rabbit polyclonal antibody exhibited good specificity.In conclusion,the polyclonal antibody was prepared which specifically recognized the PDCoV N proteins.The results provided some references for the subsequent exploration of PDCoV N protein function and laid a foundation for establishing a diag-nostic method for PDCoV.

To explore the application effect of host restriction factors(HRFs)in the development of antiviral gene drugs,this study based on the"common pathway of viral infection"and"host innate immunity HRFs",the genes of three antiviral HRFs,namely cholesterol-25-hydroxylase(CH25H),interferon-induced transmembrane protein(IFITM3)and interferon-stimulated gene 15(ISG15)were fused and expressed using pCK vectors to construct antiviral gene drugs.The fusion expression gene sequence CH25H-IFITM3-ISG15(C Ⅱ)was constructed by linking the gene cod-ing sequences of these three HRFs through the cleavage peptide coding sequence.Subsequently,the C Ⅱ sequence was amplified by PCR,ligated to the pCK expression vector,and the recombinant plasmid was transformed,identified,and extracted to obtain a candidate biodrug based on the DNA expression system,which was named pCK-CⅡ.Then,the recombinant plasmid was transfected in-to HEK 293T cells,and the expression of three antiviral proteins was successfully detected by Western blot.To clarify the antiviral effect of pCK-C Ⅱ at the cellular level,pCK-C Ⅱ was trans-fected into HEK 293 cells and BHK-21 cells,respectively.Twenty-four hours later,the BHK-21 cells were infected with vesicular stomatitis virus expressing green fluorescent protein(VSV-GFP),12 h later,the cells were observed under a fluorescence microscope and detected by flow cy-tometry;HEK 293 cells were inoculated with H3N2 subtype influenza virus,and the expression of H3N2 subtype influenza virus nuclear proteins was detected after 12 h using Western blot.The re-sults showed that transient transfection of pCK-C Ⅱ plasmid could significantly reduce the fluores-cence level of cells and the expression of nuclear protein of H3N2 subtype influenza virus in infec-ted cells.These results indicated that pCK-C Ⅱ had an inhibitory effect on the infection of VSV-GFP and H3N2 subtypes of influenza viruses after transient transfection of cells.

This study aims to screen epitope antigens targeting the receptor binding domain(RBD)of porcine epidemic diarrhea virus(PEDV)based on its amino acid sequence(GenBank accession number:AKN45969.1),prepare PEDV RBD polyclonal antibody,and perform their identification.Bioinformatics analysis software was used to predict the potential antigenic epitopes of PEDV RBD and sequence comparison with porcine coronavirus strains was performed,the selected dominant antigen epitopes were then conjugated with keyhole limpet hemocyanin(KLH),to synthesize pep-tides directly and immunize mice to generate specific antibody,Western blot technique and indirect immunofluorescence assay were utilized to identify the specificity of the antibodies,and indirect ELISA method was further applied to determine the antibody potency.Results showed the selected PEDV RBD dominant epitope sequence shared 100％similarity with 18 other PEDV strains,while exhibiting low sequence similarity with 11 TGEV strains(27.8％—29.3％)and 16 PDCoV strains(10.5％—13.4％),indicating good epitope conservation.Western blot showed that the specificity of the prepared peptide antibody specifically recognized the PEDV RED protein overexpressed in Ex-pi293F cells and overexpressed in baculovirus system,and at the same time,the antibody was still able to detect the PEDV S protein expressed in PEDV-infected Vero cells at a 1∶2 000 dilution,while it did not react with TGEV-and PDCoV-infected ST cells,indicating that the good specificity of the peptide antibody.ELISA revealed that the potency of specific antibodies in mouse serum could reach up to 1∶25 600.The above results indicate that bioinformatics techniques were suc-cessfully utilized to predict antigenic epitopes of PEDV RBD protein,and specific PEDV RBD pep-tide antibodies were prepared.

Objective: To investigate the correlation between magnetic resonance imaging-based vertebral bone quality (VBQ) score and screw loosening after dynamic pedicle screw fixation with polyetheretherketone (PEEK) rods, and evaluate its predictive value. Methods: A retrospective analysis was conducted on the patients who underwent dynamic pedicle screw fixation with PEEK rods from March 2017 to June 2022. Data on age, sex, body mass index, hypertension, diabetes, hyperlipidemia history, long-term smoking, alcohol consumption, VBQ score, L1–4 average Hounsfield unit (HU) value, surgical fixation length, and the lowest instrumented vertebra were collected. Logistic regression analysis was employed to assess the relationship between VBQ score and pedicle screw loosening (PSL). Results: A total of 24 patients experienced PSL after surgery (20.5%). PSL group and non-PSL group showed statistical differences in age, number of fixed segments, fixation to the sacrum, L1–4 average HU value, and VBQ score (p < 0.05). The VBQ score in the PSL group was higher than that in the non-PSL group (3.56 ± 0.45 vs. 2.77 ± 0.31, p < 0.001). In logistic regression analysis, VBQ score (odds ratio, 3.425; 95% confidence interval, 1.552–8.279) were identified as independent risk factors for screw loosening. The area under the receiver operating characteristic curve for VBQ score predicting PSL was 0.819 (p < 0.05), with the optimal threshold of 3.15 (sensitivity, 83.1%; specificity, 80.5%). Conclusion The VBQ score can independently predict postoperative screw loosening in patients undergoing lumbar dynamic pedicle screw fixation with PEEK rods, and its predictive value is comparable to HU value.