Objective: To identify an optimal back-flush solution for leukocyte-depleted filters that maximizes peripheral blood mononuclear cell (PBMC) recovery with high viability, long-term storage stability, and sterility of the harvested residues, thereby providing a clinically translatable strategy. Methods: Three sterile bag-packaged solutions—Saline, Solvent, and Hanks' balanced salt solution (HBSS)—were used to back-flush randomly assigned leukocyte-depleted filters. Nucleated cell recovery rate and viability of the harvested residues were compared. The optimal solution identified was applied to an expanded sample set. PBMC viability and yield were evaluated after 1h vs 48h storage of the residues. PBMCs isolated from the residues were cryopreserved in liquid nitrogen for 1 month, followed by post-thaw comparisons of viability and T-cell expansion capacity. Results: The Solvent group achieved the highest and most consistent nucleated cell recovery rate. Post-flush recovery rate from filters after 400 mL whole blood processing was (21.3±1.6)% for the Solvent group, significantly higher than Saline group (19.2±6.3)% and HBSS group (11.2±5.0)%, with residues from all groups maintaining viability >90%. No biologically significant difference in residue viability was observed between 48h vs 1h storage groups (93.3±2.3)% vs (95.7±1.8)%). PBMC recovery rates from residues showed no statistical difference between 48h vs 1h storage groups [(48.2%±9.5%)vs (40.41%±8.35%), P>0.05], with (17.7±2.6)×10 cells. After 1-month cryopreservation and 10-day expansion, PBMCs isolated from 48-hour-stored residues retained (91.2±3.2)% viability and achieved a (61.9±15.9)-fold expansion. Conclusion: The bag-packaged Solvent, as a back-flush solution, enables sterile acquisition of leukocyte-depleted filter residues through closed-system tubing connections. These residues maintained PBMC viability and recovery rates after 48h storage at 2℃-8℃, with post-cryopreservation (1-month liquid nitrogen) viability and expansion capacity remaining stable. This protocol complies with blood bank regulatory criteria, addresses the concerns about the infectious window period in cell therapy raw materials, and provides a clinically translatable strategy for PBMC-based applications.

OBJECTIVES: To characterize the biological properties of double-negative T (DNT) cells isolated from leukoreduction filter residues. METHODS: Leukoreduction filters containing residues from 400 mL whole blood units (n=6) were collected from a blood center. Filters were back-flushed with normal saline, and the eluate was concentrated to obtain leukoreduction filter residues. Leukocytes in the residues were counted by dual-fluorescence staining. DNT cells were then isolated from the residues using antibody-mediated adsorption and density gradient centrifugation. Both cryopreserved and fresh unstimulated DNT cells derived from the residues were subjected to in vitro culture. Following culture, cells were assessed for expansion fold, viability, immunophenotype, differentiation status, and cytotoxicity against target cells using dual-fluorescence staining and flow cytometry, with comparisons made to DNT cells derived from whole blood. RESULTS: The leukocyte recovery rate achieved through reverse flushing of the leukocyte reduction filter was (41.9±14.7)%. Compared to whole blood, the DNT cell starting material obtained from filter residues showed no significant difference in total T-cell content (P>0.05). However, the viability and purity of the resulting DNT cell starting materials were significantly lower (both P<0.05). After 17 days of culture, DNT cells from filter residues and whole blood showed no significant differences in expansion fold, immunophenotype, differentiation status, or cytotoxicity toward target cells (all P>0.05). However, the viability of DNT cells from residues was significantly lower than that of whole blood-derived DNT cells [(86.0±4.2)% vs. (92.2±1.2)%, P<0.05]. After thawing (post 3 or 15 days of cryopreservation) and 17 days of culture, DNT cell starting materials from residues showed comparable immunophenotype, expansion fold, and differentiation status to their non-cryopreserved counterparts from the same source (all P>0.05). However, the viability of DNT cells cryopreserved for 3 days [92.4% (91.8%, 92.8%)] and the cytotoxicity against target cells of those cryopreserved for 15 days [91.3% (89.4%, 95.1%)] were significantly higher than those of non-cryopreserved DNT cells [87.8% (82.0%, 89.0%) and 70.9% (67.3%, 80.2%), respectively] (P<0.05). CONCLUSIONS DNT cells derived from leukoreduction filter residues exhibited highly comparable characteristics to those from whole blood in terms of expansion, purity, differentiation, and biological potency. Furthermore, their biological activity post-cryopreservation and revival remained largely similar to non-cryopreserved cells. These findings suggest that leukoreduction filter residues represent a promising alternative source of starting material for manufacturing off-the-shelf, allogeneic DNT cell therapeutics.

Objective:To investigate the application value of laparoscopic natural orifice specimen extraction surgery (NOSES) based on purse-string suture for sigmoid colon and upper rectal cancer.Methods:The retrospective and descriptive study was conducted. The clinicopathological data of 14 patients undergoing laparoscopic NOSES based on purse-string suture for sigmoid colon and upper rectal cancer in The First Affiliated Hospital of Sun Yat-sen University from October 2022 to June 2023 were collected. There were 8 males and 6 females, aged (56±10)years. Observation indicators: (1) surgical conditions; (2) postoperative conditions; (3) follow-up. Measurement data with normal distribution were represented as Mean± SD. Count data were described as absolute numbers. Results:(1) Surgical conditions. All patients underwent laparoscopic NOSES based on purse-string suture for sigmoid colon and upper rectal cancer successfully, without conversion to open surgery. The operation time of 14 patients was (162±32)minutes, and the volume of intraoperative blood loss was (22±12)mL. (2) Postoperative conditions. Time to postoperative first out-of-bed activity, time to postoperative first flatus, time to postoperative first drinking, time to postoperative initial liquid food intake, duration of postoperative hospital stay of 14 patients were (1.6±0.7)days, (2.1±0.6)days, (2.4±0.6)days, (3.8±1.0)days, (6.0±0.9)days, respectively. None of patient had perioperative complications such as postoperative anastomotic leakage and bleeding. (3) Follow-up. All 14 patients were followed up for (9.7±1.9)months. There was no postoperative recurrence, metastasis or death in 14 pati-ents.Conclusion:The laparoscopic NOSES based on purse-string suture can be used for sigmoid colon and upper rectal cancer, which is safe and feasible.

Objective: To investigate the design of free perforator flap, and the efficacy of utilizing perforator flaps for Ⅳ degree Ischia-sacral ulcer treatment. Methods: From January 2010 to October 2016, 18 patients with Ⅳ degree ischia-sacral ulcer were treated. The surface area of the sacral tail ranged from 4 cm×5 cm to 8 cm ×12 cm.Doppler sonography was used to detect potential perforator.All defects were repaired with free perforator flaps, designed based on the size and shape of the wound. The flap size ranged from 6 cm×11 cm to 9 cm×15 cm. Results: One perforator flap went dehiscence after surgery, repaired by V-Y flap. All the rest of perforator flaps survived well, after 3-24 months follow-up. Flap texture and appearance was good, no ulcer reoccurred. Conclusions The free perforator flap is a simple technique.It does not need to tracethe trunk of vessels, and it does not cause major morbidities to the buttocks. Therefore, it is one of the ideal ways to repair Ⅳ degree Ischia-sacral ulcer.

Objective: To explore the effects of combined transplantation of the rat Schwann cells and fibroblasts (Fbs) on the nerve regeneration of denervated perforator flaps in rats and the mechanism. Methods: (1) Fbs were isolated from the trunk of 2 Sprague-Dawley (SD) rats embryos of 14-16 days′ pregnancy and cultured, and the morphology of the cells was observed. The third passage of cells were used for subsequent experiments. The protein expressions of fibronectin and Ephrin-B2 were observed by immunohistochemical method. The mRNA expression of Ephrin-B2 was detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction (n=3). (2) Schwann cells were isolated from the bilateral sciatic nerves and brachial plexus nerves of 45 SD rats born for 1-3 days and cultured, and the morphology of the cells was observed. The third passage of cells were used for subsequent experiments. The rate of S100 positive cells was detected by immunofluorescence method and flow cytometer, with sample numbers of 9 and 3 respectively. (3) In Dulbecco′s modified Eagle medium (DMEM) high glucose medium, 1 mL Fbs and 1 mL Schwann cells both in the concentration of 1×10⁵ cells/mL were co-cultured as Schwann cells+ Fbs co-culture group, and 2 mL Schwann cells in the concentration of 1×10⁵ cells/mL were cultured alone as Schwann cells alone culture group, with 5 wells in each group. The clusters of Schwann cells in the two groups were observed and counted under inverted phase contrast microscope at post culture hour (PCH) 6 and 24 respectively. The clusters of Schwann cells in Schwann cells+ Fbs co-culture group were observed by immunofluorescence method at PCH 24 too. The protein expressions of EphB2, Sox2, and N-cadherin in Schwann cells of two groups at PCH 24 were detected by Western blotting (n=20). (4) Totally 100 8-week-old male SD rats were selected, and an in situ replanted peritoneal denervated perforator flap was made in each rat. According to the random number table, the rats were divided into simple flap group, Fbs alone transplantation group, Schwann cells alone transplantation group, Schwann cells+ Fbs co-transplantation group, with 25 rats in each group. Flaps of rats in Fbs alone transplantation group and Schwann cells alone transplantation group were injected with 0.4 mL Fb and 0.4 mL Schwann cells respectively (2×10⁶ cells each). Flaps of rats in Schwann cells+ Fbs co-transplantation group were injected with 0.4 mL Fbs and Schwann cells mixed cells (totally 2×10⁶ cells, cell number ratio: 1∶1), and flaps of rats of simple flap group were injected with the same volume of DMEM high glucose medium. On post injection day (PID) 2, 5, 7, 9, and 14, 5 rats in each group were selected respectively according to the random number table. The flap tissue was collected, and the number, diameter, and arrangement of regenerated nerves were observed by immunofluorescence method. Data were processed with completely random designed t test, analysis of variance for repeated measurement, t test, and Bonferroni correction. Results: (1) The third passage of cells isolated and cultured from the rat embryo trunks were uniform in size and shape, long spindle-shaped, with a large proportion of nuclei. Strong positive expressions of fibronectin and Ephrin-B2 protein in cells were observed, and the mRNA expression of Ephrin-B2 was 0.004 1±0.000 8. The cells were identified as Fbs. (2) After 5 days of culture, the primary cells isolated from the sciatic nerves and brachial plexus nerves of neonatal rats were elongated in cell bodies and grew in nest, fence, or vortex-like shape. The third passage of cells were detected by immunofluorescence method and flow cytometer, and the corresponding S100 positive cell rates were (95.9±1.0)% and (95.8±1.1)% respectively. The cells were identified as Schwann cells. (3) At PCH 6 and 24, the cluster numbers of Schwann cells in Schwann cells+ Fbs co-culture group were significantly higher than those of Schwann cells alone culture group (t=6.500, 10.614, P<0.01). At PCH 24, the Schwann cells in Schwann cells+ Fbs co-culture group aggregated into clusters, Fbs dispersed around the Schwann cell clusters, and the protein expressions of EphB2, N-cadherin, and Sox2 in Schwann cells were significantly higher than those in Schwann cells alone culture group (t=2.975, 19.717, 11.159, P<0.05 or P<0.01). (4) On PID 2, a small number of scattered, disordered, short, and thin nerve fibers were observed in the flap tissue of rats in the four groups. From PID 5 to 14, the number of nerve fibers in the flap tissue of rats of Schwann cells+ Fbs co-transplantation group increased gradually, and the nerve fibers were with long diameter and arranged orderly. The number of nerve fibers in the flap tissue of rats of Schwann cells alone transplantation group increased, but the nerve fibers were with short diameter and arranged disorderly, and the number was smaller than that of Schwann cells+ Fbs co-transplantation group. In simple flap group and Fbs alone transplantation group, the nerve fibers in the flap tissue of rats gradually degenerated with gradually decreased number or even disappeared. Conclusions The combined transplantation of Fbs and Schwann cells in rats can regulate Schwann cells migration and clustering by activating Ephrin/Eph-Sox2-N-cadherin signaling pathway, thus promoting the orderly nerve regeneration of denervated perforator flaps in rats.

Objective To explore the effects of compound Danshen dripping pills on the expression levels of micro RNA-1 (miR-1) and inflammatory factors in serum of patients with acute myocardial infarction (AMI) after percutaneous coronary intervention (PCI). Methods A total of 180 AMI patients admitted to Mindong Hospital of Ningde City from March 2017 to October 2018 were divided into a compound Danshen dripping pills group and a conventional western medicine treatment group, 90 cases in each group. According to the disease situations of all the patients, they needed to undergo PCI treatment, after the intervention, in the conventional western medicine treatment group, aspirin enteric-coated tablet (metformin hydrochlorid) was given as the basic anti-coagulation medicine and in the compound Danshen dripping pill group, on the basic treatment, 10 Danshenn pills each time, 3 times a day, were orally applied. Both groups were evaluated for efficacy after 2 months of continuous treatment. Echocardiography was used to detect the patients' cardiac functions; the changes of the expression levels of serum miR-1, interleukins (IL-1, IL-6), tumor necrosis factor-α (TNF-α), and myocardial troponin I (cTnI) and creatine kinase isoenzyme (CK-MB) in the two groups before and after treatment were observed. Results After treatment in both groups, the left ventricular ejection fraction (LVEF) and cardiac output index (CI) were significantly higher than those before treatment, while the expression of miR-1 and serum IL-1, IL-6, TNF-α, cTnI and CK-MB were lower than those before treatment, the LVEF and CI in the compound Danshen dripping pill treatment group were obviously higher than those in the conventional western medicine treatment group [LVEF: 0.60±0.08 vs. 0.56±0.08, CI (L·min-1·m-2): 6.02±0.34 vs. 4.91±1.50, both P < 0.05], the expression level of miR-1 and serum IL-1, IL-6, TNF-α, cTnI and CK-MB in the compound Danshen dripping pill group were lower than those in the conventional western medicine treatment group [miR-1 (2-ΔΔCt): 0.69±0.17 vs. 0.85±0.22, IL-1 (μg/L): 59.20±18.67 vs. 68.31±23.69, IL-6 (μg/L): 20.36±1.87 vs. 25.38±2.39, TNF-α (μg/L): 28.65±1.63 vs. 31.86±2.92, cTnI (μg/L):3.12±0.88 vs. 4.03±0.97, CK-MB (U/L): 29.18±10.52 vs. 34.28±10.21, all P < 0.05]. Conclusion Compound Danshen dripping pills can reduce serum the expression levels of miR-1 and inflammatory cytokines in patients with AMI after PCI.

Objective: To investigate the feasibility of repairing soft tissue defects of extremities with the perforator flap based on the first perforating artery of profunda artery. Methods: From January 2015 to December 2018, 9 patients with soft tissue defects of extremities were treated in the Department of Burns and Plastic Surgery, Affiliated Hospital of Zunyi Medical University. 5 males and 4 females; aged 29-48 years, average 36 years old. The causes of injury were traffic accident injury (4 cases), heavy object crush injury (3 cases) and mechanical injury (2 cases). There were 3 cases of soft tissue defect of upper limb, 6 cases of soft tissue defect of lower extremity, 2 cases of tendon and nerve rupture, 2 cases of fracture, 1 case of tendon and 1 case of bone exposure. All soft tissue defects were repaired with the perforator flap based on the first perforating artery of Profunda artery. After debridement, The range of soft tissue defect was 10 cm×5 cm-16 cm×10 cm. The intraoperative flap area was 12 cm×6 cm-18 cm×11 cm. The vascular anastomosis was performed in the area, the flap covered the wound surface and the donor site directly pulled the suture or grafted skin. Results: All flaps survived completely and there were no complications such as vascular crisis. Follow-ups were performed in various forms for 6 to 12 months. These flaps were satisfactory in appearance and function and had a good texture. Conclusions The perforator flap based on the first perforating artery of profunda artery can repair the defects of the limbs in the soft group. The donor site of the flap is hidden, the amount of tissue available is relatively large and the repair effect is good.

Objective: To investigate the influence of tumor necrosis factor-related apoptosis-inducing ligant (TRAIL) deficiency on mice colitis and the gut microbiota composition by inclding the expermental colitis model in tumor necrosis factor-related apoptosis-inducing ligand gene knockout (TRAIL^-/-) mice. Methods: C57BL/6 TRAIL^-/- mice and wild type (WT) mice were selected and assigned into TRAIL^-/- control group (eight mice), TRAIL^-/- colitis group (16 mice), WT control group (eight mice) and WT colitis group (16 mice). The mice of two colitis groups were oral administrated with 3.5% dextran sulphate sodium (DSS) in drinking water for seven consecutive days to induce experimental colitis model. The severity of colitis was evaluated by clinical appearance and histopathological examination. The colonic tissue samples of mice were collected and microbiota profile was analyzed by 16S rDNA sequencing method. USEARCH software and R language were used to analyze the difference of gut microbiota among TRAIL^-/- control group, TRAIL^-/- colitis group, WT control group and WT colitis group. T test and Mann-Whitney U test were used for statistical analysis. Results: After modeling, the disease activity index (DAI) of WT colitis mice and TRAIL^-/- colitis mice both gradually increased over time. Furthermore, compared with colitis mice, TRAIL^-/- colitis mice developed body weight loss, diarrhea and hemafecia earlier. On the seventh day after modeling, the percentage of body weight loss of TRAIL^-/- colitis mice and WT colitis mice was (28.98±2.84)% and (17.87±3.70)%, respectively; and the difference was statistically significant (t=9.53, P<0.01). The length of colon of TRAIL^-/- colitis mice was shorter than that of WT colitis mice ((4.63±0.28) cm vs. (6.02±0.41) cm), and the difference was statistically significant (t=11.20, P<0.01). The DAI of TRAIL^-/- colitis mice was higher than that of WT colitis mice (3.00±0.00 vs. 2.32±0.05), and the difference was statistically significant (t=54.40, P<0.01). The histological score of TRAIL^-/- colitis mice was higher than that of WT colitis mice (6.19±0.25 vs. 3.87±0.22), and the difference was statistically significant (t=27.87, P<0.01). Under the microscope, colonic mucosal epithelial injury, crypt structure destruction and inflammatory cell infiltration were more obvious in TRAIL^-/- colitis mice than in WT colitis mice. The alpha diversity of colonic flora was more significant in TRAIL^-/- colitis group compared with that of WT colitis group. At the family level, the relative richness of Deferribacteraceae, Ruminococcaceae, Rikenellaceae, F16 and Paraprevotellaceae significantly increased in TRAIL^-/- colitis group, but the relative richness of Enterococcaceae obviously reduced ((19.839±19.991)% vs. (7.224±11.241)%, (3.564±2.543)% vs.(2.861±3.821)%, (0.123±0.066)% vs. (0.068±0.049)%, (0.032±0.033)% vs. (0.006±0.011)%, (0.153±0.098)% vs. (0.062±0.054)% and (0.013±0.027)% vs. (0.054±0.121)%, respectively; U=51, 69, 53, 35, 49 and 69, respectively; P<0.01 and 0.05, respectively). In addition, at the genus level the relative richness of Oscillospira, Mucispirillum and Cytophaga in TRAIL^-/- colitis group remarkably elevated, and the relative richness of Enterococcus significantly decreased ((2.363±2.147)% vs. (1.813±2.847)%, (19.839±19.991)% vs. (7.223±11.241)%, (0.104±0.153)% vs. (0.046±0.069)% and (0.076±0.049)% vs. (0.135±0.074)%, respectively; U=70, 51, 66 and 65, respectively; P <0.05 and 0.01, respectively). Conclusion TRAIL deficiency aggravate DSS-induced colitis, and increase the alpha diversity of colonic microbiota in colitis mice.

Objective? To investigate the status quo of depression and cognitive function of the elderly in aged care institutions in the urban area of Hangzhou, analyze the influencing factors of cognitive function and its correlation with depression, so as to provide reference for the design of later intervention measures. Methods? A total of 130 elderly people from 3 aged care institution of 3 districts of Hangzhou were recruited by convenience sampling method from March to May 2018. Montreal Cognitive Assessment and the Chinese version of Geriatric Depression Scale were applied to assess the cognitive function and depression symptoms of the elderly in aged care institutions. Logistic regression analysis was used to explore the influencing factors of cognitive function, and Pearson correlation analysis was applied to analyze the correlation between depression and cognitive function in elderly people in aged care institutions. Results? The incidence of depression in the elderly was 24.62% (32/130) and the incidence of mild cognitive impairment was 89.23% (116/130). The results of Logistic regression analysis showed that gender (OR=5.379,95%CI: 1.152-25.109), age (OR=1.129,95%CI:1.008-1.266) and educational level (OR=0.353,95%CI: 0.193-0.645) entered the model. Pearson correlation analysis showed that there was no significant correlation between depression level and cognitive function in the elderly in aged care institutions (P＞ 0.05). Conclusions? Depression and cognitive status of the elderly people in aged care institutions are in urgent need of attention. It is suggested that an intervention that focuses on reducing the incidence of depression and cognitive impairment should be provided to improve the quality of life of the elderly people.

Objective To discuss the therapeutic effect of free perforator flap of the humeral back and the healing of the wound after the removal of the malignant tumor.Methods From January,2012 to June,2016,12 cases were treated as soft tissue tumors on shoulder,including 8 cases of skin juga fibrosarcoma,3 cases of basal cell carcinoma,squamous cell carcinoma of the skin in 1 case.Preoperative using doppler ultrasound probe design perforator flap to expand resection,intraoperative cut edge basal tag frozen pathological examination without taking skin flap after the tumor invasion,according to the wound and wear the appropriate adjustment design of perforator flap.Followed-up to observe recurrence,flaps or ulcers,and the texture of the flap and the feel of the flap.All patients were followed-up regularly.Results All 12 patients were followed-up for 6-48 months.The flaps were all alive.The tumor did not relapse,and the flap was not swollen.The texture was consistent with the surrounding tissue.There was no ulceration of the flap.No obvious depression deformity.The outlook of flaps was satisfied,but the feeling was not.The doror sites were closed directly.Conclusion Adjacent using humeral back free perforators flap to repair the defect after tumor resection on shoulder is an easy operation.It is one of the ideal flaps to repair a malignant tumor on the back of the shoulder.