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 perform transient transfection of recombinant human Factor X(rhFX) into HEK293 cells,to optimize transfection parameters,to develop a high-yield in vitro expression system for rhFX production,and to assess the biological activity of expressed rhFX. Methods: The eukaryotic expression vector pcDNA3. 1-EGFP-FX was constructed by inserting the F10 gene into pcDNA3. 1-EGFP and subsequently transfected into HEK293cells to validate the transfection system efficiency. The recombinant expression vector pcDNA3. 1-FX was generated through ligation of the F10 gene fragment with pcDNA3. 1, followed by liposome-mediated transfection into HEK293 cells. The expression of rhFX in the cell supernatant was analyzed by Western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Transfection conditions were systematically optimized,and rhFX concentration was quantified by enzyme-linked immunosorbent assay(ELISA). The coagulation bioactivity of rhFX was evaluated through prothrombin time(PT) assay and chromogenic substrate method. Results: rhFX was successfully expressed in the supernatant of HEK293 cells. rhFX was successfully expressed in the supernatant of HEK293 cells. The highest expression level of rhFX was achieved on the third day after transfection when the cell density was 80% and the ratio of plasmid DNA to polyethyleneimine(PEI) transfection reagent was 1 ∶ 2.Triplicate ELISA measurements demonstrated a maximum rhFX concentration of 5. 20 ng/mL in the supernatant.The prothrombin time(PT) of rhFX-containing supernatant was significantly shorter(P 50) of etoxaban was determined to be 1. 449 nmol/L using the chromogenic substrate method based on rhFX,which was in the same order of magnitude as the reported 0. 78 nmol/L in the literature. Conclusion HEK293cells successfully express biologically active recombinant human Factor X(rhFX) protein,laying a foundation for advancing the development of rhFX-based therapeutics.

Objective:To evaluate the efficacy of purified donor CD34 positive hematopoietic stem cell (CD34 + cell) infusion in patients with poor graft function (PGF) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods:The clinical data of 25 patients with PGF who underwent donor CD34 + cell sorting and infusion at the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences between September 2019 and March 2023, were retrospectively analyzed. The cohort included 19 haploidentical and 6 HLA-matched cases. CD34 + cells were purified using immunomagnetic beads for therapeutic infusion. The purification efficiency was evaluated based on the purity and recovery rate of CD34 + cells. Clinical outcomes were assessed by hematopoietic recovery, overall survival (OS) rate, and the incidence of graft-versus-host disease (GVHD) . Results:The median total number of CD34 + cells was 2.64 (0.82-6.53) × 10 8 before purification and 2.22 (0.48-5.68) ×10 8 after purification, with a median recovery rate of 78.37% (58.48%-115.72%) . After infusion of purified CD34 + cells, 8 of 10 patients (80.0%) with poor neutrophil engraftment achieved recovery (absolute neutrophil count ≥ 0.5×10 9/L) , with a median time to recovery of 21 (10-40) days. And 15 of 21 patients (71.4%) with poor platelet engraftment achieved recovery (platelet count ≥ 20×10 9/L) , with a median time to recovery of 15 (13-38) days. Only 3 patients (12.0%) developed GVHD after the infusion of purified CD34 + cells, including 2 cases of grade I acute GVHD and 1 case of limited chronic GVHD. With a median follow-up of 14.47 (0.23-41.63) months, the overall OS rate after CD34 + cell infusion was (65.63± 8.28) %. Seventeen patients survived, with a median survival time of 19.07 (0.23-41.63) months. Conclusion:Purification of CD34 + cells using immunomagnetic beads is effective, and the infusion of these purified donor CD34 + cells can safely and effectively improve PGF after allo-HSCT.

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 evaluate the efficacy of purified donor CD34 positive hematopoietic stem cell (CD34 + cell) infusion in patients with poor graft function (PGF) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods:The clinical data of 25 patients with PGF who underwent donor CD34 + cell sorting and infusion at the Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences between September 2019 and March 2023, were retrospectively analyzed. The cohort included 19 haploidentical and 6 HLA-matched cases. CD34 + cells were purified using immunomagnetic beads for therapeutic infusion. The purification efficiency was evaluated based on the purity and recovery rate of CD34 + cells. Clinical outcomes were assessed by hematopoietic recovery, overall survival (OS) rate, and the incidence of graft-versus-host disease (GVHD) . Results:The median total number of CD34 + cells was 2.64 (0.82-6.53) × 10 8 before purification and 2.22 (0.48-5.68) ×10 8 after purification, with a median recovery rate of 78.37% (58.48%-115.72%) . After infusion of purified CD34 + cells, 8 of 10 patients (80.0%) with poor neutrophil engraftment achieved recovery (absolute neutrophil count ≥ 0.5×10 9/L) , with a median time to recovery of 21 (10-40) days. And 15 of 21 patients (71.4%) with poor platelet engraftment achieved recovery (platelet count ≥ 20×10 9/L) , with a median time to recovery of 15 (13-38) days. Only 3 patients (12.0%) developed GVHD after the infusion of purified CD34 + cells, including 2 cases of grade I acute GVHD and 1 case of limited chronic GVHD. With a median follow-up of 14.47 (0.23-41.63) months, the overall OS rate after CD34 + cell infusion was (65.63± 8.28) %. Seventeen patients survived, with a median survival time of 19.07 (0.23-41.63) months. Conclusion:Purification of CD34 + cells using immunomagnetic beads is effective, and the infusion of these purified donor CD34 + cells can safely and effectively improve PGF after allo-HSCT.

Objective:To construct a machine-learning model for predicting the progression of pancreatic cystic lesions (PCLs) based on clinical and CT features, and to evaluate its predictive performance in internal/external testing cohorts.Methods:Baseline clinical and radiological data of 200 PCLs in 177 patients undergoing abdominal thin slice enhanced CT examination at Peking Union Medical College Hospital from July 2014 to December 2022 were retrospectively collected. PCLs were divided into progressive and non-progressive groups according to whether the signs indicated for surgery by the guidelines of the European study group on PCLs were present during three-year follow-up. 200 PCLs were randomly divided into training (150 PCLs) and internal testing cohorts (50 PCLs) at the ratio of 1∶3. 15 PCLs in 14 patients at Jinling Affiliated Hospital of Medical School of Nanjing University from October 2011 to May 2020 were enrolled as external testing cohort. The clinical and CT radiological features were recorded. Multiple feature selection methods and machine-learning models were implemented and combined to identify the optimal machine-learning model based on the 10-fold cross-validation method. Receiver operating characteristics (ROC) curve was drawn and area under curve (AUC) was calculated. The model with the highest AUC was determined as the optimal model. The optimal model's predictive performance was evaluated on testing cohort by calculating AUC, sensitivity, specificity and accuracy. Permutation importance was used to assess the importance of optimal model features. Calibration curves of the optimal model were established to evaluate the model's clinical applicability by Hosmer-Lemeshow test.Results:In training and internal testing cohorts, the progressive and non-progressive groups were significantly different on history of pancreatitis, lesions size, main pancreatic duct diameter and dilation, thick cyst wall, presence of septation and thick septation (all P value <0.05) In internal testing cohort, the two groups were significantly different on gender, lesion calcification and pancreatic atrophy (all P value <0.05). In external testing cohort, the two groups were significantly different on lesions size and pancreatic duct dilation (both P<0.05). The support vector machine (SVM) model based on five features selected by F test (lesion size, thick cyst wall, history of pancreatitis, main pancreatic duct diameter and dilation) achieved the highest AUC of 0.899 during cross-validation. SVM model for predicting the progression of PCLs demonstrated an AUC of 0.909, sensitivity of 82.4%, specificity of 72.7%, and accuracy of 76.0% in the internal testing cohort, and 0.944, 100%, 77.8%, and 86.7% in the external testing cohort. Calibration curved showed that the predicted probability by the model was comparable to the real progression of PCLs. Hosmer-Lemeshow goodness-of-fit test affirmed the model's consistency with actual PCLs progression in testing cohorts. Conclusions:The SVM model based on clinical and CT features can help doctors predict the PCLs progression within three-year follow-up, thus achieving efficient patient management and rational allocation of medical resource.

Objective:To investigate whether murine peroxidase reductase-5(mPRDX5)has anti-tumor activity in mice,so as to further confirm the anti-tumor activity and mechanism of recombinant peroxidase reductase-5.Methods:High purity mPRDX5 was obtained by heterologous expression and purification in vitro.Pancreatic cancer Pan02 cells were inoculated subcutaneously on the left axillary back of mice to establish a tumor bearing mouse model.Mice were randomly divided into PBS(solvent control)group,GEM(gemcitabine)50.0 mg/kg group and mPRDX5 10.0 mg/kg group,with 10 mice in each group,and the tumor related indexes were detected in mice.Results:Compared with PBS group,weight of tumor-bearing mice in GEM group was decreased obviously,while weight of mPRDX5 group was increased to a certain extent.Tumor growth was good in PBS group,according to tumor volume,com-pared with PBS group,tumor growth inhibition rates in D7 were 87.07%in GEM group and 52.82%in mPRDX5 10.0 mg/kg group,re-spectively;according to tumor weight,compared with PBS group,GEM group and mPRDX5 10.0 mg/kg group had tumor growth inhibi-tion rates of 95.39%and 48.33%in D7,respectively.Polarization state of macrophages in tumor tissues of mice in PBS group and mPRDX5 group was analyzed,and it was found that compared with PBS group,M1 macrophages expressing CD86 in tumor tissues of mice in mPRDX5 group were significantly increased,while M2 macrophages expressing CD206 were significantly decreased.Conclu-sion:mPRDX5 has significant anti-pancreatic cancer activity in mice,and the activity is exerted by promoting M1-type polarization of macrophages in the tumor microenvironment.

BACKGROUND:Stem cell transplantation is a new way to prevent and cure intervertebral disc degeneration.However,whether the transplanted stem cells can survive,proliferate,differentiate,and restore the function of nucleus pulposus cells after transplantation,is the key and difficult point to overcome. OBJECTIVE:To explore the effects of Bushenhuoxue decoction on survival,proliferation,and nucleus pulposus-like differentiation of adipose-derived stem cells. METHODS:A Transwell chamber was used to construct a co-culture model of human adipose-derived stem cells and human degenerative nucleus pulposus cells.The experiment was divided into control group,model group,drug-containing serum group,and drug-free serum group.Except for the control group,the co-culture system of other groups was treated with 50 μmol/L tert-butyl hydrogen peroxide for 24 hours.The drug-containing serum group and drug-free serum group were treated with DMEM low-glucose complete culture medium containing drug-containing serum of Bushenhuoxue decoction or drug-free serum with 20％volume fraction for 48 hours.The sublayer adipose-derived stem cells were taken.Toluidine blue staining was used to detect proteoglycan synthesis levels.Real-time PCR method was used to detect mRNA expression of type Ⅱ collagen,proteoglycan and SRY-box transcription factor 9.The protein expression of SOX9 was detected by western blot assay.Lactate dehydrogenase assay was used to detect cytotoxicity.Flow cytometry was used to detect reactive oxygen species,and β-galactosidase staining was used to detect cell senescence. RESULTS AND CONCLUSION:(1)Compared with the control group,the proportion of necrotic cells in the model group increased;toluidine blue staining became lighter,and the expression levels of type Ⅱ collagen,proteoglycan,SOX9 mRNA and SOX9 protein decreased(P<0.05).Compared with the model group,the drug-containing serum of Bushenhuoxue decoction could significantly reduce cell injury and promote the expression of type Ⅱ collagen,proteoglycan,SOX9 mRNA,and SOX9 protein(P<0.05),but the improvement in the drug-free serum group was not significant(P>0.05).(2)Compared with the control group,the contents of cytotoxicity,reactive oxygen species,and cell senescence in the model group were significantly increased.Compared with the model group,the microenvironment of the coculture system was significantly improved by drug-containing serum of Bushenhuoxue decoction(P<0.05),while drug-free serum had no significant effect on the microenvironment of the co-culture system(P>0.05).(3)The results show that Bushenhuoxue decoction can promote the survival,proliferation,and nucleus pulposus-like differentiation of adipose-derived stem cells.

Objective:To investigate the clinical efficacy of butylphthalide combined with hyperbaric oxygen therapy on post-stroke cognitive impairment in patients with acute ischemic stroke.Methods:A total of 90 patients with post-stroke cognitive impairment who were hospitalized within 72 hours of onset in Suining County People's Hospital from December 2019 to November 2020 were included in this study. They were randomly divided into a control group and an observation group ( n = 45/group). The control group was given conventional treatment and the observation group was given butylphthalide combined with hyperbaric oxygen therapy in addition to conventional treatment. The National Institutes of Health Stroke Scale score, Montreal Cognitive Assessment score, and Activities of Daily Living score were compared between the two groups before and after treatment. Results:Before treatment, there were no significant differences in the National Institutes of Health Stroke Scale score, Montreal Cognitive Assessment score, and Activities of Daily Living score between the two groups (all P > 0.05). At 14 days and 1 month after surgery, the National Institutes of Health Stroke Scale scores in the observation group were (4.02 ± 2.18) points and (3.21 ± 2.03) points, which were significantly lower than (5.21 ± 2.24) points and (4.62 ± 2.68) points in the control group ( t =2.55, 2.81, both P < 0.05). At 1 and 3 months after treatment, the Montreal Cognitive Assessment score in the observation group were (19.79 ± 5.67) points and (23.69 ± 2.67) points, which were significantly higher than (16.88 ± 5.12) points and (19.74 ± 2.29) points in the control group ( t = 2.56, 7.53, both P < 0.05). At 1 and 3 months after treatment, Activities of Daily Living scores in the observation group were (54.85 ± 5.69) points and (74.38 ± 4.98) points, which were significantly higher than (46.78 ± 6.24) points and (63.21 ± 5.24) points in the control group ( t = 6.41, 9.76, both P < 0.05). Conclusion:Butylphthalide combined with hyperbaric oxygen therapy for the treatment of post-stroke cognitive impairment in patients with acute ischemic stroke can alleviate neurologic deficits, and improve cognitive function and the ability of daily life.

By combing out the historical development logic of sports and health integration and the current development achievement, the underlying logic of sports and health integration is described systematically in multiple dimensions, and the prominent dilemmas in development are analyzed as follows: lack of residents' health literacy, excessive dependence on medical treatment, low bridging of talent team construction, lack of the guidance of funds centralization, insufficient financial support, imperfect management system and mechanism, and unspecific departmental collaboration.The strategies for solving the problems are as follows: strengthening the integration of sports science and clinical medicine, consolidating the technical integration of sports prescription and clinical standardization process, further improving the business integration of exercise intervention and clinical treatment, and enhancing the industrial integration of sports products and medical treatment.