BACKGROUND:Exosomes derived from mesenchymal stem cells play pivotal roles in cell communication and epigenetic regulation due to their low immunogenicity and targeted delivery effects,and have been clinically applied in the treatment of various diseases.OBJECTIVE:To review the isolation,purification,identification methods,and application progress of mesenchymal stem cell-derived exosomes,and to facilitate the development of large-scale preparation techniques and clinical translation of mesenchymal stem cell-derived exosomes.METHODS:The Chinese search terms"exosome,mesenchymal stem cells,isolation,purification,characterization,clinical application"and the English search terms"exosome,extracellular vesicles,mesenchymal stem cells,isolation,characterization,application"were used to search the literature published before September 2024 in CNKI,PubMed,and Web of Science databases.Articles with poor relevance to the topic,outdated,or duplicated content were excluded,and finally,109 articles were included for review.RESULTS AND CONCLUSION:(1)This paper reviews recent methods for isolating and purifying exosomes,comparing the characteristics of ultracentrifugation,ultrafiltration,size-exclusion chromatography,polymer precipitation,immunoaffinity,microfluidic methods,and other novel approaches based on their underlying principles.(2)Methods for identifying exosomes can be categorized into physical and biochemical analyses,characterizing exosomes based on their shape,size,and characteristic proteins.(3)Mesenchymal stem cell-derived exosomes have broad applications in multiple fields such as medical aesthetics,wound repair,and cancer treatment,due to their immune-regulatory properties and ability to cross biological barriers.(4)The clinical translation of exosomes faces challenges due to their complex structure,lack of universal isolation techniques,and poor stability,making it difficult to achieve in a short period of time.

ObjectiveThis paper aims to investigate the material basis of the anti-inflammatory efficacy and mechanism of action of Bushen Tongdu prescription （BSTDP）. MethodsThe chemical components of BSTDP and its blood-absorbed components in vivo were systematically identified by using ultra-performance liquid chromatography-linear ion trap-electrostatic field orbitrap high-resolution mass spectrometry （UPLC-LIT-Orbitrap-MS）. Network pharmacology was employed to screen blood-absorbed bioactive components and potential targets of this formula. A protein-protein interaction （PPI） network of core targets was constructed to conduct enrichment analysis. Molecular docking was further utilized to verify the binding affinity between key components and targets. The inflammatory model was established and verified in vivo by using a transgenic zebrafish Tg （mpx： GFP）. At three days post-fertilization （3 dpf）， larvae of zebrafish were randomly assigned to blank group， model group， positive drug dexamethasone acetate group （75 μmol·L^-1）， and BSTDP groups with low， medium， and high doses （500， 1 000， and 2 000 mg·L^-1）. The distribution and quantity of neutrophils in the yolk sac region were observed under a fluorescence microscope. The mRNA expression levels of key genes in the toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor kappa-B （NF-κB） signaling pathway and inflammatory factors including interleukin （IL）-1β， IL-6， and tumor necrosis factor-α （TNF-α） were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsA total of 120 chemical components were identified in BSTDP， among which 26 original components were confirmed by using serum pharmacochemical methods. A total of 227 common targets linking rheumatoid arthritis （RA） and the blood-absorbed components were screened by network pharmacology. It is suggested that pseudobrucine， vomicine， sinapine， rehmannioside， cinnamyl alcohol glycoside， and methylephedrine exert anti-inflammatory effects by acting on core targets including protein kinase B1 （Akt1）， signal transducer and activator of transcription 3 （STAT3）， tumor necrosis factor （TNF）， TLR4， mitogen-activated protein kinase 14 （MAPK14）， and phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit α （PIK3CA）， thereby modulating multiple signaling pathways such as TLR4 and NF-κB. In vivo verification in zebrafish demonstrates that the maximum tolerable concentration of Bushen Tongdu Formula is 2 000 mg·L^-1. Compared to those in the blank group， zebrafish in the model group showed a significantly higher number of neutrophils in the yolk sac region （P<0.01） and rising mRNA levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β （P<0.01）. Compared to that in the model group， the number of neutrophils was significantly reduced in BSTDP groups with medium and high doses， as well as the dexamethasone acetate group （P<0.05， P<0.01）. There was no statistically significant difference in the low dose group. The mRNA expression levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β were significantly down-regulated （P<0.05， P<0.01）. ConclusionThis paper identifies the material basis of the efficacy of BSTDP， demonstrating that the formula can exert an anti-inflammatory effect through the TLR4/MyD88/NF-κB signaling pathway. The results provide scientific experimental evidence for its further clinical application.

ObjectiveThis paper aims to investigate the material basis of the anti-inflammatory efficacy and mechanism of action of Bushen Tongdu prescription （BSTDP）. MethodsThe chemical components of BSTDP and its blood-absorbed components in vivo were systematically identified by using ultra-performance liquid chromatography-linear ion trap-electrostatic field orbitrap high-resolution mass spectrometry （UPLC-LIT-Orbitrap-MS）. Network pharmacology was employed to screen blood-absorbed bioactive components and potential targets of this formula. A protein-protein interaction （PPI） network of core targets was constructed to conduct enrichment analysis. Molecular docking was further utilized to verify the binding affinity between key components and targets. The inflammatory model was established and verified in vivo by using a transgenic zebrafish Tg （mpx： GFP）. At three days post-fertilization （3 dpf）， larvae of zebrafish were randomly assigned to blank group， model group， positive drug dexamethasone acetate group （75 μmol·L^-1）， and BSTDP groups with low， medium， and high doses （500， 1 000， and 2 000 mg·L^-1）. The distribution and quantity of neutrophils in the yolk sac region were observed under a fluorescence microscope. The mRNA expression levels of key genes in the toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor kappa-B （NF-κB） signaling pathway and inflammatory factors including interleukin （IL）-1β， IL-6， and tumor necrosis factor-α （TNF-α） were detected by Real-time quantitative polymerase chain reaction （Real-time PCR）. ResultsA total of 120 chemical components were identified in BSTDP， among which 26 original components were confirmed by using serum pharmacochemical methods. A total of 227 common targets linking rheumatoid arthritis （RA） and the blood-absorbed components were screened by network pharmacology. It is suggested that pseudobrucine， vomicine， sinapine， rehmannioside， cinnamyl alcohol glycoside， and methylephedrine exert anti-inflammatory effects by acting on core targets including protein kinase B1 （Akt1）， signal transducer and activator of transcription 3 （STAT3）， tumor necrosis factor （TNF）， TLR4， mitogen-activated protein kinase 14 （MAPK14）， and phosphatidylinositol-4，5-bisphosphate 3-kinase catalytic subunit α （PIK3CA）， thereby modulating multiple signaling pathways such as TLR4 and NF-κB. In vivo verification in zebrafish demonstrates that the maximum tolerable concentration of Bushen Tongdu Formula is 2 000 mg·L^-1. Compared to those in the blank group， zebrafish in the model group showed a significantly higher number of neutrophils in the yolk sac region （P<0.01） and rising mRNA levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β （P<0.01）. Compared to that in the model group， the number of neutrophils was significantly reduced in BSTDP groups with medium and high doses， as well as the dexamethasone acetate group （P<0.05， P<0.01）. There was no statistically significant difference in the low dose group. The mRNA expression levels of TLR4， MyD88， NF-κB， TNF-α， IL-6， and IL-1β were significantly down-regulated （P<0.05， P<0.01）. ConclusionThis paper identifies the material basis of the efficacy of BSTDP， demonstrating that the formula can exert an anti-inflammatory effect through the TLR4/MyD88/NF-κB signaling pathway. The results provide scientific experimental evidence for its further clinical application.

BACKGROUND:Exosomes derived from mesenchymal stem cells play pivotal roles in cell communication and epigenetic regulation due to their low immunogenicity and targeted delivery effects,and have been clinically applied in the treatment of various diseases.OBJECTIVE:To review the isolation,purification,identification methods,and application progress of mesenchymal stem cell-derived exosomes,and to facilitate the development of large-scale preparation techniques and clinical translation of mesenchymal stem cell-derived exosomes.METHODS:The Chinese search terms"exosome,mesenchymal stem cells,isolation,purification,characterization,clinical application"and the English search terms"exosome,extracellular vesicles,mesenchymal stem cells,isolation,characterization,application"were used to search the literature published before September 2024 in CNKI,PubMed,and Web of Science databases.Articles with poor relevance to the topic,outdated,or duplicated content were excluded,and finally,109 articles were included for review.RESULTS AND CONCLUSION:(1)This paper reviews recent methods for isolating and purifying exosomes,comparing the characteristics of ultracentrifugation,ultrafiltration,size-exclusion chromatography,polymer precipitation,immunoaffinity,microfluidic methods,and other novel approaches based on their underlying principles.(2)Methods for identifying exosomes can be categorized into physical and biochemical analyses,characterizing exosomes based on their shape,size,and characteristic proteins.(3)Mesenchymal stem cell-derived exosomes have broad applications in multiple fields such as medical aesthetics,wound repair,and cancer treatment,due to their immune-regulatory properties and ability to cross biological barriers.(4)The clinical translation of exosomes faces challenges due to their complex structure,lack of universal isolation techniques,and poor stability,making it difficult to achieve in a short period of time.

Objective: To investigate the trends in incidence and mortality of lung cancer in Huangpu District, Shanghai Municipality from 2002 to 2019, so as to provide the evidence for formulating lung cancer prevention and control measures. Methods: Data of lung cancer incidence and mortality among residents in Huangpu District from 2002 to 2019 were collected through the Shanghai Cancer Registration and Reporting Management System. The crude incidence and mortality of lung cancer was calculated, and standardized by the data from the Chinese Fifth National Population Census in 2000 (Chinese-standardized rate) and the Segi's world standard population in 1960 (world-standardized rate). The trends in incidence and mortality of lung cancer among residents by age and gender were evaluated using annual percent change (APC). Results: A total of 12 965 cases of lung cancer were reported in Huangpu District from 2002 to 2019, and the crude incidence rate was 80.66/105, the Chinese-standardized incidence rate was 34.54/105, and the world-standardized incidence rate was 31.30/105, all showing upward trends (APC=4.588%, 2.933% and 3.247%, all P<0.05). A total of 10 102 deaths of lung cancer were reported, and the crude mortality rate was 62.30/105, showing an upward trend (APC=0.959%, P<0.05); the Chinese-standardized mortality was 25.93/105, and the world-standardized mortality was 22.05/105, both showing downward trends (APC=-1.282% and -1.263%, both P<0.05). The crude incidence and mortality rates of lung cancer in males were higher than those in females (101.39/105 vs. 60.52/105, 85.45/105 vs. 39.87/105, both P<0.05). The crude incidence and mortality rates of lung cancer showed upward trends with age (both P<0.05), reaching their peaks in the age groups of 80-<85 years (341.37/105) and 85 years or above (355.97/105), respectively. Conclusions The incidence of lung cancer showed an upward trend, while the mortality showed a downward trend in Huangpu District from 2002 to 2019. Elderly men were the high-risk group for lung cancer incidence and mortality.

Background::The increasing incidence of inflammatory bowel disease (IBD) presents significant medical and societal challenges. A well-designed IBD database is crucial for both epidemiological studies and clinical management. However, inconsistencies between regional databases hinder cross-institutional and international research, especially between Eastern and Western societies.Methods::We developed a new IBD database, the 301 IBD database, integrating the IBD clinical characteristics from the Penn IBD database (USA) and the latest IBD guidelines and consensus and clinical practices of the Chinese PLA General Hospital (PLAGH). We applied this database to analyze clinical data of IBD inpatients at PLAGH from 2008 to 2023.Results::The 301 IBD database contains 490 items in 6 sections including demographic characteristics, personal history, clinical phenotype, disease activity, laboratory tests and examinations, and treatment. Features of the 301 IBD database include inpatient focus, biochemical indicators and opportunistic infection focus, and more about ulcerative colitis (UC)-associated complications. Single-center analysis revealed an increasing hospitalization trend, from 2.35% in 2008 to 3.94% in 2023. We found that the clinical characteristics of our UC inpatients are predominantly male (62.5%), extensive lesions (55.1%), low usage of biologics (4.1%), and a high incidence of UC-CRC (3.0%). The clinical characteristics of CD inpatients included male predominance (68.39%), early onset age (35.43 ± 14.75-year-old), and high rate of surgery (25.81%).Conclusion::The 301 IBD database, integrating Eastern and Western clinical data, provides a valuable tool for IBD clinical research. Future international, multicenter collaborations are expected to further enhance its utility.

Objective:To observe the changes of central visual acuity and extracentral visual acuity in eyes with non-arteritic central retinal artery occlusion (NA-CRAO).Methods:A retrospective clinical study. From January 1, 2017 to December 31, 2024, 140 patients (140 eyes) diagnosed with NA-CRAO through ophthalmic examination at Department of Ophthalmology of First People's Hospital of Xianyang City were included in the study. All affected eyes underwent best corrected visual acuity (BCVA), visual field, intraocular pressure, fundus color photography, optical coherence tomography (OCT), and fluorescein angiography (FFA) examinations. After a clear diagnosis, conservative treatment such as reducing intraocular pressure, relieving spasms, and dilating blood vessels should be given immediately. Simultaneously, intravenous and/or arterial thrombolysis therapy should be administered based on the patient's overall condition. Under the same treatment conditions as other treatments, 33 eyes were treated with hyperbaric oxygen therapy within 24 hours after seeking medical attention. The changes in central visual acuity (BCVA) and peripheral visual acuity of the affected eye one month after treatment were observed. BCVA improvement of ≥ 1 line was defined as the increase of no light sensitivity to light sensitivity or above, and the increase of light sensitivity to 0.01 or above. The visual acuity outside the center was determined by the 0 ° axis in front of the eyeball at eye level, and was 10 ° outside visual acuity on the temporal side. Multivariate analysis using logistic regression analysis.Results:Among the 140 cases (140 eyes), there were 84 males (84 eyes) and 56 females (56 eyes). The mean age was (63.89±10.78) years. The duration of illness from the onset of symptoms to the time of diagnosis was 48 (2-720) hours. 6, 1, 14, 47, 41, 16, and 15 eyes were diagnosed with BCVA without light perception, uncertain light perception, manual/anterior, digital/anterior, 0.01-0.10, and ≥ 0.10, respectively. FFA examination revealed delayed arm retinal circulation time and filling of the retinal artery trunk to the peak, with changes in the "arterial front" observed in 126 eyes. OCT examination showed extensive edema and unclear structure in the inner layer of the retina in all patients. Out of 140 eyes, 122 were treated with intravenous thrombolysis and 4 with arterial thrombolysis; 14 eyes did not receive thrombolytic therapy. After treatment, 38 eyes (27.1 %) showed an improvement of BCVA ≥ 1; 67 eyes (47.9%) did not show an improvement in BCVA, and the affected eye had a BCVA of approximately 0.6 without light perception; 17 eyes (12.1 %) showed improvement in peripheral vision, and the peripheral vision of the affected eyes ranged from 0.01 to 0.1, all of whom were patients undergoing intravenous thrombolysis, and prior to treatment, this group of patients had complete blindness in the coarse side visual field of the Amsler grid, and their out of center visual acuity could not be measured. Among the 33 eyes treated with hyperbaric oxygen therapy, 24 eyes (72.7%) showed an increase in BCVA after treatment; 9 eyes did not improve, among which 4 eyes (12.1%) showed improvement in out of center visual acuity. Among the 107 eyes that did not receive hyperbaric oxygen therapy, 49 eyes (45.8%) showed an increase in BCVA after treatment. There was no improvement in 58 eyes (54.2%), among which 13 eyes (12.1%) showed an improvement in out of center visual acuity. The results of logistic regression analysis showed that intravenous thrombolysis and hyperbaric oxygen therapy were independent predictive factors for the improvement of central and extra central visual acuity ( P<0.05). Conclusions:Hyperbaric oxygen therapy within 24 hours of seeking medical attention for patients with NA-CRAO disease course ≤ 1 month has a significant effect on the recovery of central and extra central vision. Intravenous thrombolysis and hyperbaric oxygen therapy are independent predictive factors for the improvement of central and extra central vision.

Objective:To investigate the prevalence of flight fatigue among helicopter flying personnel and analyze its contributors in order to provide data for related interventions.Methods:A cross-sectional study was conducted among 404 helicopter flying personnel between October 8, 2021 and July 31, 2022. Data was collected using a self-designed questionnaire, involving the demography of these subjects, sleep-related factors, flight fatigue, perceived causes of fatigue and coping strategies. The Pittsburgh Sleep Quality Index (PSQI), National Aeronautics and Space Administration Task Load Index (NASA-TLX) and Modified Fatigue Impact Scale (MFI-20) were used to assess sleep quality, mental workload, and levels of flight fatigue over the past month. The total scores of MFI-20 were compared across demographic groups, and correlations with PSQI and NASA-TLX scores were analyzed. Multiple linear regression was performed to identify the determinants of flight fatigue.Results:①Demography: among the 404 helicopter flying personnel, 92.8% (375/404) were pilots and 7.2% (29/404) navigators. As for years of service, 41.6% (168/404) served less than 5 years, while 58.4% (236/404) served more than 5 years. 37.9% (153/404) had a family history of insomnia. 18.8% (76/404) did not habitually nap, 68.9% (226/328) napped for ≤30 min, 31.1% (102/328) napped over 30 min, and 18.3% (74/404) had insomnia over the past month. As for helicopter flying personnel, 75.5% (305/404) reported experiencing fatigue, with 69.1% (279/404) attributing it to flight-related factors and 51.5% (208/404) using coffee as a countermeasure.②Scale scores: the total score of PSQI was [5 (3, 7)], while the highest daytime dysfunction score was [1(0, 2)]. The total score of NASA-TLX was [39.19 (26.57, 51.97)], and the effort score was the highest [10.31(5.07, 14.60)]. The total score of MFI-20 averaged (47.28±14.88), with the mental fatigue score being the highest [(10.03±4.42)]. ③Comparisons of MFI-20 total scores: flying personnel with ≤5 years of flying experience had higher MFI-20 total scores than those with >5 years, and those with a family history of insomnia had higher scores than those without ( t=3.35, 2.44, P=0.001, 0.015). Individuals with insomnia over the past month had higher scores than non-insomniacs ( t=3.33, P=0.001). Significant differences in MFI-20 scores were observed based on nap duration ( F=19.95, P<0.001). Non-nappers had higher scores than those napping for ≤30 min ( P=0.005). Flying personnel who napped for >30 min had higher scores than those did not ( P=0.043) or napped for ≤30 min ( P<0.001). ④Correlation analysis: the total score of MFI-20 was positively correlated with sleep quality, sleep latency, sleep disturbances, hypnotic medications, daytime dysfunction, and the total score of PSQI ( r=0.118-0.226, all P<0.05), but negatively with sleep duration ( r=-0.136, P=0.006). The total score of MFI-20 was positively correlated with mental demand, physical demand, and the total score of NASA-TLX ( r=0.119, 0.168, 0.184, P=0.017, 0.001, <0.001). ⑤Multiple linear regression analysis: the determinants of flight fatigue included aircraft types ( B=-4.956, 95% CI:-8.124--1.788), nap duration ( B=3.693, 95% CI: 1.267-6.119), sleep latency ( B=2.371, 95% CI: 0.229-4.513), sleep duration ( B=-7.383, 95% CI:-10.008--4.758), daytime dysfunction ( B=5.003, 95% CI: 2.967-7.039) and physical workload ( B=0.611, 95% CI: 0.324-0.898). Conclusions:Helicopter flying personnel are vulnerable to flight fatigue, which is strongly linked to sleep quality and mental workload. It is crucial to address flying personnel′s self-perceived fatigue, care about fatigue manifestations across aircraft types, and implement targeted interventions to improve sleep quality and reduce mental workload.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Background::The increasing incidence of inflammatory bowel disease (IBD) presents significant medical and societal challenges. A well-designed IBD database is crucial for both epidemiological studies and clinical management. However, inconsistencies between regional databases hinder cross-institutional and international research, especially between Eastern and Western societies.Methods::We developed a new IBD database, the 301 IBD database, integrating the IBD clinical characteristics from the Penn IBD database (USA) and the latest IBD guidelines and consensus and clinical practices of the Chinese PLA General Hospital (PLAGH). We applied this database to analyze clinical data of IBD inpatients at PLAGH from 2008 to 2023.Results::The 301 IBD database contains 490 items in 6 sections including demographic characteristics, personal history, clinical phenotype, disease activity, laboratory tests and examinations, and treatment. Features of the 301 IBD database include inpatient focus, biochemical indicators and opportunistic infection focus, and more about ulcerative colitis (UC)-associated complications. Single-center analysis revealed an increasing hospitalization trend, from 2.35% in 2008 to 3.94% in 2023. We found that the clinical characteristics of our UC inpatients are predominantly male (62.5%), extensive lesions (55.1%), low usage of biologics (4.1%), and a high incidence of UC-CRC (3.0%). The clinical characteristics of CD inpatients included male predominance (68.39%), early onset age (35.43 ± 14.75-year-old), and high rate of surgery (25.81%).Conclusion::The 301 IBD database, integrating Eastern and Western clinical data, provides a valuable tool for IBD clinical research. Future international, multicenter collaborations are expected to further enhance its utility.