ObjectiveTo construct a stable monoclonal human embryonic kidney 293 （HEK293） cell line expressing recombinant human coagulation factor Ⅶ （rhFⅦ） and evaluate the expression level and procoagulant bioactivity of rhFⅦ. MethodsThe plasmid pCDNA3.1-EGFP-FⅦ was transfected into HEK293 cells to verify the effectiveness of the transfection system. The plasmid pCDNA3.1-FⅦ was transfected into HEK293 cells， and monoclonal stable transfected cell lines were selected using geneticin （G418）. The transcription of the FⅦ gene was identified by reverse transcription polymerase chain reaction （RT-PCR）. The expression level of rhFⅦ in the supernatant of the monoclonal stable transfected cell line was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. The concentration of rhFⅦ was determined by enzyme-linked immunosorbent assay （ELISA）， and the procoagulant activity of rhFⅦ was measured by human coagulation factor Ⅶ potency assay. ResultsHEK293 cells transfected with pcDNA3.1-EGFP-FⅦ showed green fluorescence， indicating that rhFⅦ was successfully expressed in the supernatant of HEK293 cells after transient transfection with pcDNA3.1-FⅦ. The monoclonal stable transfected cell line was obtained by G418 screening. RT-PCR identified that the FⅦ gene was integrated into the genome of the monoclonal stable transfected cell line. The cell viability was good as detected by Cell Counting Kit-8， and a single band of rhFⅦ was obtained by purification of the cell supernatant. The highest rhFⅦ expression was （1.27±0.09） mg/L， and the highest procoagulant activity was （380.29±13.80）%. ConclusionThe monoclonal HEK293 cell lines which can express rhFⅦ protein efficiently and stably with excellent procoagulant bioactivity is successfully screened.

Objective To develop a scale-down model in the purification process for effectively evaluating the lifetime of chromatography medium in the production process of recombinant Mycobacterium tuberculosis fusion protein(EC), providing technical basis and optimization strategies for industrial production.MethodsBased on the principle of linear scaling, a 156-fold scale-down column model was constructed. The reliability of the scale-down model was evaluated by comparing key performance indicators such as chromatogram, protein recovery rate, removal rate of host protein, removal rate of residual antibiotics, removal rate of exogenous DNA, and electrophoretic purity between the scale-down model and commercial production scale. On this basis, the scale-down model was used to perform 40 cycles of chromatography purification, and the chromatographic performance data of the target product was analyzed to evaluate the long-term effectiveness of the chromatography medium.ResultsThe chromatographic performance indicators of the scale-down model and commercial production scale, including chromatogram features, protein recovery rate, host protein removal rate, and electrophoretic purity, were highly consistent with excellent data stability. After 40 cycles, the protein content, purity, residual host protein,antibiotic activity, and residual exogenous DNA of the target product all met the quality standards. The column efficiency(HETP, As) of the chromatography column met the process requirements.ConclusionThis study successfully verified the effectiveness of the scale-down model and determined that the chromatography medium lifetime is tentatively set at 40 cycles, providing reliable technical support for industrial production.

Background: Disorders of the blood-brain barrier (BBB) arising from diabetes mellitus are closely related to diabetic encephalopathy. Previous research has suggested that neuron-glia antigen 2 (NG2)-glia plays a key role in maintaining the integrity of the BBB. However, the mechanism by which NG2-glia regulates the diabetic BBB remains unclear. Methods: Type 2 diabetes mellitus (T2DM) db/db mice and db/m mice were used. Evans-Blue BBB permeability tests and transmission electron microscopy techniques were applied. Tight junction proteins were assessed by immunofluorescence and transmission electron microscopy. NG2-glia number and signaling pathways were evaluated by immunofluorescence. Detection of matrix metalloproteinase-9 (MMP-9) in serum was performed using enzyme-linked immunosorbent assay (ELISA). Results: In T2DM db/db mice, BBB permeability in the hippocampus significantly increased from 16 weeks of age, and the structure of tight junction proteins changed. The number of NG2-glia in the hippocampus of db/db mice increased around microvessels from 12 weeks of age. Concurrently, the expression of MMP-9 increased in the hippocampus with no change in serum. Sixteen- week-old db/db mice showed activation of the Wnt/β-catenin signaling in hippocampal NG2-glia. Treatment with XAV-939 improved structural and functional changes in the hippocampal BBB and reduced MMP-9 secretion by hippocampal NG2-glia in db/db mice. It was also found that the upregulation of β-catenin protein in NG2-glia in the hippocampus of 16-week-old db/db mice was significantly alleviated by treatment with XAV-939. Conclusion The results indicate that NG2-glia can lead to structural and functional disruption of the diabetic BBB by activating Wnt/β-catenin signaling, upregulating MMP-9, and degrading tight junction proteins.

Objective:To analyze by bioinformatics the association between the expression of membrane-associated tyrosine/threonine protein kinase 1(PKMYT1)in glioma with its prognostic value,biological function,drug sensitivity,gene mutation,and immune infiltration.Methods:The differential expression of PKMYT1 was analyzed based on the Chinese Glioma Genome Atlas database(CGGA)and the Cancer Genome Atlas database(TCGA).Pathways likely to be enriched for PKMYT1 were predicted by gene ontology analysis(GO)and gene set enrichment analysis(GSEA).PKMYT1 was subjected to Pearson correlation and gene set variation analysis(GSVA)with cell cycle-related genes and gene sets.The survival prognosis,gene mutation,drug sensitivity and immune infiltration were further analyzed for PKMYT1 high and low expression groups of glioma patients.Results:PKMYT1 was significantly highly expressed in WHO high-grade glioma(P<0.000 1),IDH wild-type glioma(P<0.05),and glioblastoma(P<0.000 1).Overall survival(OS)of patients in the PKMYT1 low expression group was significantly higher than that of the high expression group(P<0.05).Cox regression analysis showed that PKMYT1 expression level was an independent prognostic factor for OS(P<0.05).GO and GSEA analyses showed that the gene sets co-expressed with PKMYT1 were mainly enriched in signaling pathways such as cell cycle,DNA replication and DNA damage repair.Pearson correlation and GSVA analyses showed that the expression of PKMYT1 was significantly and positively correlated with the cell cycle-related genes,gene sets and cell cycle checkpoint genes(P<0.01).Drug sensitivity analysis revealed that patients in the PKMYT1 high expression group had high sensitivity osimertinib,dabrafenib,carmustine and cediranib(P<0.05).Mutation analysis revealed that the IDH1 gene had a higher mutation frequency in the PKMYT1 low expression group.The results of immune infiltration analysis showed that PKMYT1 expression was significantly positively correlated with glioma stroma score(r=0.13,P<0.001),immune score(r=0.11,P<0.01)and ESTIMATE score(r=0.13,P<0.001);and was significantly positively correlated with immune cell infiltration level of regulatory T(Treg)cells and M2-type macrophages(P<0.05).Conclusion:Patients with high PKMYT1 expression have a poorer prognosis,and the mechanism may be related to tumor immune infiltration and cell cycle regulation.PKMYT1 is expected to be a potential target for the diagnosis and treatment of glioma.

Objective:To investigate the key technical aspects of laparoscopic anti-reflux surgery (LARS) in complex gastroesophageal reflux disease (GERD) with hiatal hernia (HH) and evaluate the feasibility and efficacy of reoperation.Methods:A retrospective analysis was conducted on 28 patients with complex GERD treated at Zhejiang Provincial People's Hospital from Feb 2020 to May 2024. Preoperative examinations were recorded, and surgical videos were reviewed to reconstruct operative time, critical intraoperative steps, complications, and management techniques. Postoperative follow-up via telephone and outpatient visits assessed symptom relief, complications, and medication use.Results:All 28 patients (4 robotic-assisted and 24 conventional laparoscopic surgeries) successfully underwent LARS, with an operative duration of (152.6±10.3) minutes and a postoperative hospital stay of (4.0±1.9) days. Large HH 9 cases, intraoperative bleeding 6 cases, pleural rupture 3 case, and esophageal perforation 1 case, preoperative diagnoses included short esophagus 2 cases and 7 redo surgeries. The overall recurrence rate was 11%. Postoperative complications occurred in 14%. The redo surgeries group achieved 71% symptom resolution. At 1-12 months of follow-up, 82% of patients were asymptomatic, and 82% discontinued proton pump inhibitor therapy.Conclusions:Complex scenarios requiring specialized techniques in LARS increase surgical difficulty and risks. Standardized management of the hernia sac, hiatal repair, neurovascular protection, identification of anatomical landmarks in reoperations, selection of biological mesh, and adhesiolysis may reduce recurrence rates and complication risks.

Objective:To investigate the key technical aspects of laparoscopic anti-reflux surgery (LARS) in complex gastroesophageal reflux disease (GERD) with hiatal hernia (HH) and evaluate the feasibility and efficacy of reoperation.Methods:A retrospective analysis was conducted on 28 patients with complex GERD treated at Zhejiang Provincial People's Hospital from Feb 2020 to May 2024. Preoperative examinations were recorded, and surgical videos were reviewed to reconstruct operative time, critical intraoperative steps, complications, and management techniques. Postoperative follow-up via telephone and outpatient visits assessed symptom relief, complications, and medication use.Results:All 28 patients (4 robotic-assisted and 24 conventional laparoscopic surgeries) successfully underwent LARS, with an operative duration of (152.6±10.3) minutes and a postoperative hospital stay of (4.0±1.9) days. Large HH 9 cases, intraoperative bleeding 6 cases, pleural rupture 3 case, and esophageal perforation 1 case, preoperative diagnoses included short esophagus 2 cases and 7 redo surgeries. The overall recurrence rate was 11%. Postoperative complications occurred in 14%. The redo surgeries group achieved 71% symptom resolution. At 1-12 months of follow-up, 82% of patients were asymptomatic, and 82% discontinued proton pump inhibitor therapy.Conclusions:Complex scenarios requiring specialized techniques in LARS increase surgical difficulty and risks. Standardized management of the hernia sac, hiatal repair, neurovascular protection, identification of anatomical landmarks in reoperations, selection of biological mesh, and adhesiolysis may reduce recurrence rates and complication risks.

Periodontitis is a critical risk factor for the occurrence and development of diabetes.Porphyromonas gingivalis may participate in insulin resistance(IR)caused by periodontal inflammation,but the functional role and specific mechanisms of P.gingivalis in IR remain unclear.In the present study,clinical samples were analysed to determine the statistical correlation between P.gingivalis and IR occurrence.Through culturing of hepatocytes,myocytes,and adipocytes,and feeding mice P.gingivalis orally,the functional correlation between P.gingivalis and IR occurrence was further studied both in vitro and in vivo.Clinical data suggested that the amount of P.gingivalis isolated was correlated with the Homeostatic Model Assessment for IR score.In vitro studies suggested that coculture with P.gingivalis decreased glucose uptake and insulin receptor(INSR)protein expression in hepatocytes,myocytes,and adipocytes.Mice fed P.gingivalis tended to undergo IR.P.gingivalis was detectable in the liver,skeletal muscle,and adipose tissue of experimental mice.The distribution sites of gingipain coincided with the downregulation of INSR.Gingipain proteolysed the functional insulin-binding region of INSR.Coculture with P.gingivalis significantly decreased the INSR-insulin binding ability.Knocking out gingipain from P.gingivalis alleviated the negative effects of P.gingivalis on IR in vivo.Taken together,these findings indicate that distantly migrated P.gingivalis may directly proteolytically degrade INSR through gingipain,thereby leading to IR.The results provide a new strategy for preventing diabetes by targeting periodontal pathogens and provide new ideas for exploring novel mechanisms by which periodontal inflammation affects the systemic metabolic state.

Objective:To summarize the experience using laparoscopic Heller myotomy combined with Dor fundoplication for the treatment of achalasia of cardia.Methods:This is a retrospective analysis. From Mar 2020 to Mar 2023 8 patients admitted at Zhejiang Provincial People's Hospital were treated surgically, including 5 males and 3 females with a mean age of (47±15) years.Results:Dysphagia was the most common preoperative symptom, followed by acid reflux. All patients underwent preoperative gastroscopy and esophageal high-resolution manometry. Most patients underwent gastrointestinal barium series,ultrasound endoscopy, and chest/abdomen/esophageal CT. All patients underwent laparoscopic Heller myotomy + Dor fundoplication, 2 cases suffered from intraoperative esophageal mucosal injury, and received intraoperative mucosal repair. Mean postoperative hospital stay was (4.38±2.23) days, and 2 cases complaining dysphagia within 1 month after surgery, treated conservatively recovered and discharged. The Eckardt score was significantly lower in all patients after surgery than before surgery ( P=0.011). Conclusion:Laparoscopic Heller myotomy combined with Dor fundoplication has good short and medium-term efficacy in the treatment of patients with achalasia of the cardia.

Objective:To improve and optimize the modified surgical strategies for patents with Simon Ⅰ and Simon Ⅱ type of gynecomastia, based on their magnetic resonance imaging (MRI) features.Methods:Clinical data of 190 patients aged 12 to 56 years (mean age 26.7±8.6) with gynecomastia who underwent modified surgeries in the Department of Plastic Surgery of Peking Union Medical College Hospital from January 2017 to January 2023 were retrospectively analyzed. The study measured breast MRI images of 44 male patients to calculate the area ratio of the glands on the transverse plane via nipple. This provided insight into the physiological structure and the tissue distribution of gynecomastia for improving the modified surgical strategies. All 190 patients were treated with enhanced liposuction and " Pull-Through and Bottom-Up" techniques. The duration of operation, amount of liposuction and amount of glandular resection, postoperative complications were recorded, and postoperative satisfaction was investigated by questionnaire surveys.Results:MRI images of the 87-side male breast showed that the average area ratio of the glands on the transverse plane via nipple was (10.9±12.5) %. The most prevalent subtype of gynecomastia was branch pattern, accounting for 46.0%. The median surgical duration of 190 patients was 95 (65-210) minutes, the median liposuction volume was 300 (50-1 400) ml, and the median glandular removal was 19.9 (1.5-157.0) g. Eighty-eight patients (46.3%) went through followed up for more than 6 months post-posterative with an overall satisfaction score of 4.68±0.53. Postoperative complications occurred in 19 sides (5.1%), and the reoperation rate was 1.3%.Conclusions:The MRI imaging of gynecomastia shows that the component of patients′ breasts are mainly fat, with a small amount of glandular tissue and the dominant subtype is branch pattern. Enhanced liposuction combined with " Pull-Through and Bottom-Up" stab incision technique can be an effective treatment for Simon grades Ⅰ and Ⅱ gynecomastia. The method results in high patient satisfaction with fewer postoperative complications.

Objective:To analyze the correlation between chronic obstructive pulmonary disease (COPD) and cognitive dysfunction.Methods:This is a case-control study. From February 2022 to October 2022, 32 COPD patients (inpatient and outpatient) from the Department of Respiratory and Critical Care Medicine and Rehabilitation Medical Center of the First Affiliated Hospital of Nanjing Medical University and 32 healthy subjects were recruited. All participants underwent a thorough evaluation, which included Montreal Assessment of Cognitive Function (MoCA), visuospatial n-back task included accuracy (ACC) and mean response time (RT), the pulmonary functions including forced vital capacity (FVC), forced expiratory volume in the first second (FEV 1), one-second rate (FEV 1/FVC) and maximum volume per minute (MVV), Health Survey Short Form (SF-36), and St. George′s Respiratory Questionnaire (SGRQ). The correlation between cognitive dysfunction and lung function, SF-36 and SGRQ in COPD patients were analyzed. Results:The prevalence of smoking, hypertension and cardiovascular disease in the two groups were significantly different (all P<0.05). MoCA score, 1-back ACC and 2-back ACC in COPD group were significantly lower than those in healthy control group [(23.86±4.50) vs (27.55±1.29) points, (76.82%±16.60%) vs (90.61%±7.40%), (67.93%±10.10%) vs (78.74%±10.38%), all P<0.001]; 2-back RT was significantly higher than that of healthy group [(316.43±108.17) vs (254.09±101.62) ms, P<0.05]; and the Physiological function (PF), physiological function (RP), emotional function (RE), energy (VT), social function (SF), physical pain (BP) in SF-36 were significantly worse than the healthy control group (all P<0.05). The MoCA score of COPD group was positively correlated with FEV 1/FVC ( r=0.501, P=0.018). The 1-back ACC was positively correlated with FEV 1 and FEV 1/FVC ( r=0.568, 0.634; both P<0.05). The 1-back RT was negatively correlated with FEV 1/FVC and MVV ( r=-0.452, -0.534; both P<0.05). The 2-back ACC was positively correlated with FEV 1/FVC ( r=0.426, P=0.048). The 2-back RT was negatively correlated with MVV ( r=-0.571, P=0.006). In COPD group, MoCA score was negatively correlated with activity, influence and total score in SGRQ ( r=-0.533, -0.466, -0.521; all P<0.05). The 1-back ACC was negatively correlated with activity, influence and total score ( r=-0.552, -0.517, -0.584; all P<0.05). The 1-back RT was positively correlated with activity, influence and total score ( r=0.430, 0.379, 0.417; all P<0.05). The 2-back ACC was negatively correlated with impact and total score ( r=-0.398, -0.412; both P<0.05). Conclusion:COPD patients have impaired cognitive function, which is mainly manifested by the decline of working memory and executive function, and is correlated with the lung function, general health condition and quality of life.