Objective: To explore the types and levels of exosomes in cryoprecipitate from donors of different blood types, genders, and age groups, and to preliminarily characterize their specific proteins. Methods: Exosomes were extracted from cryoprecipitate using the polymer precipitation method. The exosomes were identified by particle size analysis, transmission electron microscopy, and Western blot. Finally, the specific proteins CD63, CD81, CD9, Cytochrome C, Syntenin-1, and VLA-4 of exosomes were characterized using Luminex. Results: The purified exosomes exhibited a double-membrane vesicle structure under microscopy, appearing saucer-shaped or as hemispheres with a concave side. They specifically expressed the CD63 and TSG101 proteins, while the endoplasmic reticulum protein Calnexin was not expressed. The particle sizes(nm) of exosomes from blood types A, B, O, and AB were 119.6±47.5, 120.5±47.4, 133.3±54.1 and 103.1±35.3, respectively. Taking the exosomal specific protein CD63 as an example, its expression levels(pg/mL) in cryoprecipitate from female and male donors were 128.22±63.52 and 117.39±40.84, respectively; the expression(pg/mL) levels in the 20-40 and 40-60 age groups were 114.20±37.28 and 131.41±64.98, respectively; and the expression levels(pg/mL) in the four blood types A, B, O, and AB were 109.58±37.79, 127.45±44.46, 131.89±40.59, and 122.29±78.68, respectively. The expression levels of CD63 in exosomes from different blood types, genders, and age groups were similar, with no statistically significant differences (all P>0.05). The distribution of the other five exosomal-specific proteins was also comparable among the normal blood donors. Conclusion: The expression levels of exosomal-specific proteins in cryoprecipitate from normal blood donors are not associated with blood type, gender, and age group.

ABSTRCT AIM:To verify the effect of polygona-tum polysaccharide(PSP)combined with TGF-β3 in inducing the differentiation of rat tendon-derived stem cells(TDSCs)into chondrocytes by activating the TGF-β3/Smad2 pathway.METHODS:TDSCs were extracted from rat tail tendons using enzyme digestion,purified,passaged,and identified via flow cytometry.TDSCs were treated with different concentrations of PSP,and the optimal growth con-centration was determined using the CCK-8 assay.TDSCs were divided into four groups:PSP,TGF-β3,PSP+TGF-β3,and Control for differentiation induc-tion.Chondrogenic differentiation was evaluated using morphological observations,toluidine blue staining,immunofluorescence staining,and West-ern blot analysis to detect COLⅡ,SOX9,and AGG.Western blot was also used to measure the expres-sion levels of Smad2 and p-Smad2 to evaluate the activation of the TGF-β3/Smad2 pathway after chondrogenic induction.RESULTS:Flow cytometry analysis showed that TDSCs highly expressed CD90 and CD29,while CD11b and CD45 expression was low.The CCK-8 assay indicated that 5 μmol/L PSP was the optimal intervention dose.Toluidine blue staining revealed that the blue staining area in the PSP,PSP+TGF-β3,and TGF-β3 groups was larger compared to the Control group.Immunofluores-cence analysis demonstrated that COLⅡ expression was significantly increased in the PSP,TGF-β3,and PSP+TGF-β3 groups,with the highest expression in the PSP+TGF-β3 group(P＜0.05).Western blot analy-sis showed that the levels of p-Smad2/Smad2,COLⅡ,SOX9,and AGG were elevated in the PSP,TGF-β3,and PSP+TGF-β3 groups,with the highest ex-pression in the PSP+TGF-β3 group(P＜0.05).Compared to the Control group,the TGF-β3 and PSP groups also showed significantly increased expression(P＜0.05).CONCLUSION:PSP promotes the proliferation and chondrogenic differentiation of TDSCs,possibly by activating the TGF-β3/Smad2 pathway.

Objective To establish orthotopic and ectopic pancreatic cancer models in C57BL/6N mice with normal immune function using in vivo imaging technology for visual characterization.Methods Orthotopic and ectopic pancreatic cancer models were established in Kunming mice by injecting a small volume of cell suspension containing firefly luciferase-expressing Panc02-luciferase pancreatic cancer cells into the head of the pancreas or the right axillary region.In vivo imaging technology was used to optimize the modeling method and timing in Kunming mice.Subsequently,the same method was applied to C57BL/6N mice using wild-type Panc02 pancreatic cancer cells to establish orthotopic and ectopic pancreatic cancer models with intact immune function.Key parameters,including body weight,inoculation positive rate,tumor growth time,tumor volume,and pathological characteristics across different organs,were compared be-tween the orthotopic and ectopic models in C57BL/6N mice to evaluate the applicability of these models.Results Both the small animal in vivo imaging experiments in Kunming mice and the tumor growth observation in C57BL/6N mice demonstrated that the construction periods for orthotopic and ectopic pancreatic cancer models were 20 days,with survival rates exceeding 90%.The inoculation positive rates in C57BL/6N mice were 92.3%for the orthotopic model and 78.6%for the ectopic model.On day 20 post-inoculation,the tumor volumes were(117.04±109.56)mm3 for the orthotopic model and(155.68±168.73)mm3 for the ectopic model,indicating high model success rates and consistent tumor growth.HE staining revealed pathological mitotic figures and poorly differentiated tumor tissues in both models of C57BL/6N mice,with no evidence of metastasis to other organs.Conclusions Orthotopic and ectopic pancreatic cancer models in immu-nocompetent mice were successfully developed in this study,mimicking early-stage pancreatic cancer characteristics.These models pro-vide a reliable platform for screening early diagnostic biomarkers and evaluating therapeutic interventions for pancreatic cancer.

Objective:To investigate the clinical application of internal mammary perforator (IMP) as recipient vessels in immediate breast reconstruction using deep inferior epigastric perforator (DIEP) flap.Methods:The clinical data of 10 patients with early breast cancer who underwent DIEP for immediate breast reconstruction using IMP as the recipient vessels from January 2022 to December 2023 were analyzed. The number, position, and diameter of IMP, diameter of DIEP, the size of the flap, the operation time, surgical complications, cosmetic effect of breast, and patients' satisfaction were summarized and analyzed.Results:The number of IMPs was 2-3, and they were distributed in the second to the fourth ribs. The diameter of the IMP artery was (1.15±0.22) mm, and the diameter of the vein was (1.35±0.19) mm. The diameter of the DIEP was (1.55±0.28) mm, and that of its accompanying vein was (1.50±0.23) mm. The sizes of the flaps ranged from 10.0 cm×8.0 cm×3.0 cm to 12.0 cm×22.0 cm×4.0 cm, with an average of 20.5 cm×11.2 cm×2.8 cm. The weight of the flap was (389.1±51.5) g. The operation time was (574.8±68.1) min. All 10 cases of flaps survived. The reconstructed breasts were natural, soft, and symmetrical to the healthy side breasts. There was no obvious operative complication. The average BREAST-Q score of the patients was 93.5. No recurrence or metastasis was found during follow-up.Conclusions:Although technically difficult, using IMP as recipient vessels in DIEP flats for immediate breast reconstruction results in a low complication rate of the injuries in the internal mammary region. Under the premise of strictly adhering to the surgical indications, satisfactory results can be achieved, and it is safe and reliable.

Objective:To investigate the impact of the number of examined lymph nodes (ELN) on survival outcomes in gastric cancer patients with postoperative pathological stage pN3b.Methods:This retrospective cohort study included 279 pN3b gastric cancer patients who underwent D2 gastrectomy at Nanfang Hospital, Southern Medical University (September 2008 to April 2023), with 35 patients receiving combination chemotherapy and anti-PD-1 therapy (immunotherapy group) and 244 receiving adjuvant chemotherapy alone (nonimmunotherapy group). Additionally, 422 patients with pN3b from the SEER database (2005 to 2020) were collected as an external validation cohort to determine the optimal cutoff value for the number of lymph nodes examined in the nonimmunotherapy group. The primary endpoints were overall survival (OS) and recurrence-free survival (RFS) in the nonimmunotherapy group of the Nanfang Hospital cohort, stratified by whether the number of examined lymph nodes was above or below the ELN optimal cutoff value. These findings were subsequently validated in the SEER cohort.Results:The optimal ELN cutoff value (34 nodes) was determined using X-tile software and by constructing an ELN-HR fitting model with inflection point identification. In the nonimmunotherapy group, patients with ELN >34 exhibited significantly prolonged survival compared to ELN ≤34 (median OS: 25.0 (95%CI:20.5-29.5) to 17.0 (95%CI:12.7-21.3) months, P=0.004; median RFS: 19.0 (95%CI:15.6-22.4) to 13.0 (95%CI:9.5-16.5) months, P=0.048). Multivariate Cox analysis also showed ELN >34 to be an independent protective factor for both OS (HR=0.576, 95%CI: 0.397-0.836) and RFS (HR=0.701, 95%CI: 0.492-0.998). In the SEER cohort, ELN >34 was associated with a 5-month OS extension (19 to 14 months, P=0.065), with multivariate analysis supporting its independent prognostic significance (HR=0.729, 95%CI: 0.580-0.915, P=0.006). Notably, in the immunotherapy group, patients with ELN >34 ( n=30) achieved a median OS of 41 months, but the median OS had not been reached in the ELN ≤34 group ( n=5) (1 death at 48 months). Conclusion:Higher ELN (>34) correlates with improved survival in nonimmunotherapy-treated pN3b gastric cancer patients. However, in pN3b gastric cancer patients treated with immunotherapy, the optimal ELN threshold requires further exploration to determine.

ABSTRCT AIM:To verify the effect of polygona-tum polysaccharide(PSP)combined with TGF-β3 in inducing the differentiation of rat tendon-derived stem cells(TDSCs)into chondrocytes by activating the TGF-β3/Smad2 pathway.METHODS:TDSCs were extracted from rat tail tendons using enzyme digestion,purified,passaged,and identified via flow cytometry.TDSCs were treated with different concentrations of PSP,and the optimal growth con-centration was determined using the CCK-8 assay.TDSCs were divided into four groups:PSP,TGF-β3,PSP+TGF-β3,and Control for differentiation induc-tion.Chondrogenic differentiation was evaluated using morphological observations,toluidine blue staining,immunofluorescence staining,and West-ern blot analysis to detect COLⅡ,SOX9,and AGG.Western blot was also used to measure the expres-sion levels of Smad2 and p-Smad2 to evaluate the activation of the TGF-β3/Smad2 pathway after chondrogenic induction.RESULTS:Flow cytometry analysis showed that TDSCs highly expressed CD90 and CD29,while CD11b and CD45 expression was low.The CCK-8 assay indicated that 5 μmol/L PSP was the optimal intervention dose.Toluidine blue staining revealed that the blue staining area in the PSP,PSP+TGF-β3,and TGF-β3 groups was larger compared to the Control group.Immunofluores-cence analysis demonstrated that COLⅡ expression was significantly increased in the PSP,TGF-β3,and PSP+TGF-β3 groups,with the highest expression in the PSP+TGF-β3 group(P＜0.05).Western blot analy-sis showed that the levels of p-Smad2/Smad2,COLⅡ,SOX9,and AGG were elevated in the PSP,TGF-β3,and PSP+TGF-β3 groups,with the highest ex-pression in the PSP+TGF-β3 group(P＜0.05).Compared to the Control group,the TGF-β3 and PSP groups also showed significantly increased expression(P＜0.05).CONCLUSION:PSP promotes the proliferation and chondrogenic differentiation of TDSCs,possibly by activating the TGF-β3/Smad2 pathway.

Objective To establish orthotopic and ectopic pancreatic cancer models in C57BL/6N mice with normal immune function using in vivo imaging technology for visual characterization.Methods Orthotopic and ectopic pancreatic cancer models were established in Kunming mice by injecting a small volume of cell suspension containing firefly luciferase-expressing Panc02-luciferase pancreatic cancer cells into the head of the pancreas or the right axillary region.In vivo imaging technology was used to optimize the modeling method and timing in Kunming mice.Subsequently,the same method was applied to C57BL/6N mice using wild-type Panc02 pancreatic cancer cells to establish orthotopic and ectopic pancreatic cancer models with intact immune function.Key parameters,including body weight,inoculation positive rate,tumor growth time,tumor volume,and pathological characteristics across different organs,were compared be-tween the orthotopic and ectopic models in C57BL/6N mice to evaluate the applicability of these models.Results Both the small animal in vivo imaging experiments in Kunming mice and the tumor growth observation in C57BL/6N mice demonstrated that the construction periods for orthotopic and ectopic pancreatic cancer models were 20 days,with survival rates exceeding 90%.The inoculation positive rates in C57BL/6N mice were 92.3%for the orthotopic model and 78.6%for the ectopic model.On day 20 post-inoculation,the tumor volumes were(117.04±109.56)mm3 for the orthotopic model and(155.68±168.73)mm3 for the ectopic model,indicating high model success rates and consistent tumor growth.HE staining revealed pathological mitotic figures and poorly differentiated tumor tissues in both models of C57BL/6N mice,with no evidence of metastasis to other organs.Conclusions Orthotopic and ectopic pancreatic cancer models in immu-nocompetent mice were successfully developed in this study,mimicking early-stage pancreatic cancer characteristics.These models pro-vide a reliable platform for screening early diagnostic biomarkers and evaluating therapeutic interventions for pancreatic cancer.

ObjectiveTo evaluate the effects of Tongnaoyin on the blood-brain barrier status and neurological impairment in acute ischemic stroke （AIS） patients with the syndrome of phlegm-stasis blocking collaterals by dynamic contrast-enhanced magnetic resonance imaging （DCE-MRI）. MethodsA total of 63 patients diagnosed with AIS in the Jiangsu Province Hospital of Chinese Medicine from October 2022 to December 2023 were enrolled in this study. According to random number table method，the patients were assigned into a control group （32 cases） and an observation group （31 cases）. The control group received conventional Western medical treatment，and the observation group took 200 mL Tongnaoyin after meals，twice a day from day 2 of admission on the basis of the treatment in the control group. After 7 days of treatment，the patients were examined by DCE-MRI. The baseline data for two groups of patients before treatment were compared. The National Institute of Health Stroke Scale （NIHSS） score and modified Rankin Scale （mRS） score were recorded before treatment and after 90 days of treatment for both groups. The rKtrans，rKep，and rVe values were obtained from the region of interest （ROI） of the infarct zone/mirror area and compared between the two groups. ResultsThere was no significant difference in the NIHSS or mRS score between the two groups before treatment. After 90 days of treatment，the NIHSS and mRS scores declined in both groups，and the observation group had lower scores than the control group （P<0.05）. After treatment，the rKtrans and rVe in the observation group were lower than those in the control group （P<0.01）. ConclusionCompared with conventional Western medical treatment alone，conventional Western medical treatment combined with Tongnaoyin accelerates the repair of the blood-brain barrier in AIS patients，thereby ameliorating neurological impairment after AIS to improve the prognosis.

Objective: To explore the association between negative life events and smartphone addiction among middle school students, so as to provide theoretical support and practical guidance for prevention and intervention of smartphone addiction among middle school students. Methods: Using cluster sampling, 8 890 students were selected to survey from 27 junior high schools and 3 senior high schools in a district of Shenzhen in 2022 (baseline) and 2023 (followup). Data were collected through selfresigned questionnaires on basic information, the Smartphone Addiction Scale-Short Version, and the Adolescent Selfrating Life Events Checklist. Mixedeffects models were employed to analyze the association. Results: Compared to 2022, the punishment scores of middle school students in 2023 [1.00 (0.00, 6.00) and 1.00 (0.00, 6.00)] decreased (Z=4.27), while the scores of interpersonal stress, learning stress and adaptation [4.00(0.00, 8.00), 4.00(0.00, 8.00); 4.00(1.00, 8.00), 5.00(2.00, 9.00); 2.00 (0.00, 6.00), 3.00 (0.00, 7.00)] increased (Z=-3.04, -8.36, -6.80) (P<0.01). Mixedeffects models revealed a positive doseresponse relationship between negative life events and smartphone addiction (OR=1.08-1.17, P<0.01). Stepwise regression showed independent positive effects of interpersonal stress (OR=1.05), academic stress (OR=1.03), and adaptation stress (OR=1.11) on smartphone addiction (P<0.01). Subgroup analysis of nonaddicted students in 2022 confirmed persistent associations for academic stress (OR=1.03) and adaptation (OR=1.07) (P<0.01). Conclusion Negative life events exhibit a positive doseresponse relationship with smartphone addiction, particularly interpersonal stress, academic stress, and adaptationrelated events.

Objective: To explore the potential causal association between adolescent compulsive behaviour and smartphone addiction based on longitudinal data, so as to provide reference for the establishment of adolescent smartphone addiction interventions. Methods: A preliminary survey and follow-up were conducted on 8 907 middle and high school students in a district of Shenzhen in 2022 and 2023, respectively. Compulsive behaviours were measured by using the Mental Health Inventory for Middle School Students-60 Items (MMHI-60), smartphone addiction was assessed by using the Smartphone Addiction Scale-Short Version ( SAS- SV), and the associations between compulsive behaviours and smartphone addiction were analysed by using multilevel mixed-effects models and subgroup analyses. Results: Smartphone addiction detection rates among middle school students were significantly associated with genders, father s education level, mother s education level, study load subgroups, and whether or not they were single-parent families, and there were statistical differences ( χ 2=17.21-175.34, P <0.05). Students with compulsive behaviours were 2.98 times more likely to develop smartphone addiction than those without compulsive behaviours ( OR=2.98, 95%CI=2.77-3.22, P <0.05). Subgroup analysis of middle school students without smartphone addiction in the first year found that compulsive behaviours significantly predicted smartphone addiction ( OR= 1.76 , 95%CI=1.54-2.01, P <0.05). Conclusion There is a potential causal association between obsessive-compulsive behaviours and smartphone addiction in middle school students, and obsessive-compulsive behaviours in middle school students could significantly predicted the occurrence of smartphone addiction.