OBJECTIVE: To prepare clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSC) with high expression of hematopoietic supporting factors and evaluate their stem cell characteristics. METHODS: Fetal umbilical cord tissues were collected from healthy postpartum women during full-term cesarean section. Wharton's jelly was mechanically separated and hUC-MSCs were obtained by explant culture method and enzyme digestion method in an animal serum-free culture system with addition of human platelet lysate. The phenotypic characteristics of hUC-MSCs obtained by two methods were detected by flow cytometry. The differences in proliferation ability between the two groups of hUC-MSCs were identified through CCK-8 assay and colony forming unit-fibroblast (CFU-F) assay. The differences in multilineage differentiation potential between the two groups of hUC-MSCs were identified through induction of adipogenic, osteogenic, and chondrogenic differentiation. The mRNA expression levels of hematopoietic supporting factors such as SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in the two groups of hUC-MSCs were identified by real-time fluorescence quantiative PCR(RT-qPCR). RESULTS: The results of flow cytometry showed that hUC-MSCs obtained by the two methods both expressed high levels of CD73, CD90 and CD105, while lowly expressed CD31, CD45 and HLA-DR. The results of CCK-8 and CFU-F assay showed that the proliferation ability of hUC-MSCs obtained by explant culture method was better than those obtained by enzyme digestion method. The results of the triple lineage differentiation experiment showed that there was no significant difference in multilineage differentiation potential between the two grous of hUC-MSCs. The results of RT-qPCR showed that the mRNA expression levels of hematopoietic supporting factors SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in hUC-MSCs obtained by explant cultrue method were higher than those obtained by enzyme digestion method. CONCLUSION Clinical-grade hUC-MSCs with high expression levels of hematopoietic supporting factors were successfully cultured in an animal serum-free culture system.
Humans ; Mesenchymal Stem Cells/metabolism* ; Umbilical Cord/cytology* ; Cell Differentiation ; Female ; Cell Proliferation ; Cells, Cultured ; Chemokine CXCL12/metabolism* ; Angiopoietin-1/metabolism* ; Vascular Cell Adhesion Molecule-1/metabolism* ; Stem Cell Factor/metabolism* ; Flow Cytometry ; Pregnancy

Apurinic/apyrimidinic endonuclease 1(APE 1)is a multifunctional protein that plays important roles in DNA repair and regulation of gene expression.Because APE 1 is overexpressed in various cancers,it can serve as a cancer biomarker for aiding clinical diagnosis,guiding therapy,and monitoring prognosis.On this basis,a label-free fluorescent probe was designed based on the primer exchange reaction(PER)strategy for highly sensitive detection of APE 1 activity.In the absence of APE 1,the structure of catalytic hairpin(HP)was stable and could not form G-quadruplex.Therefore,the background fluorescence of this sensing system was very low due to the dissociation of thioflavin T(ThT).In the presence of APE 1,the apurinic/apyrimidinic(AP)site of HP was cleaved by APE 1 and a short nucleic acid fragment that acted as a primer to initiate PER was generated.After PER reaction,a large number of G-quadruplex were produced,which could specifically bind with ThT and resulted in significant increase of fluorescence signal.The combination of low background design of HP and PER amplification made this biosensor had high sensitivity with a detection limit(3σ)of 0.0008 U/mL.Furthermore,the primer sequence was directly generated by the cleavage of APE 1 without additional addition,which not only increased the specificity of the reaction,but also simplified the experiment procedure.Moreover,the use of label-free fluorescence signal reduced the cost of the experiment,and realized rapid detection of APE 1.Finally,this sensor was used to detect APE 1 in human serum samples with spiked recoveries of 91%-104%,proving great potential in study of biological enzyme.

Objective To identify the taste critical quality attribute and design and optimize the flavor-correcting formulation of the traditional Chinese medicine oral preparation Qingre Jiedu Oral Liquid,in order to improve its taste and enhance patient medication adherence.Methods The taste assignment method was employed to identify the taste critical quality attribute of Qingre Jiedu Oral Liquid.Based on human sensory evaluation and the standardized Euclidean distance in electronic tongue analysis,suitable types of corrigent were determined.Subsequently,under constraints such as maximum allowable dosage,solubility,and sweetness,the optimal taste formulation for the sugar-free intermediate of Qingre Jiedu Oral Liquid was determined using Box-Behnken experimental design combined with electronic tongue and human sensory evaluation results.The study was reviewed and approved by the Ethics Committee of Beijing University of Chinese Medicine(Ethics Approval Number 2020BZYLL0609).Results The quantitative score for bitter taste of Qingre Jiedu Oral Liquid accounted for 30.36%,confirming bitterness as the taste critical quality attribute requiring attention.The optimal taste formulation for the sugar-free intermediate of Qingre Jiedu Oral Liquid was determined to be 120 mg·mL?1 erythritol,12 mg·mL?1 acesulfame potassium,and 2.4 mg·mL?1 stevioside.This formulation achieved an 11.75-point improvement in sensory evaluation scores compared to the original commercially available oral liquid.Conclusion This study successfully improved the taste of Qingre Jiedu Oral Liquid and established a comprehensive strategy for flavor-correcting formulation optimization,including a method for identifying taste critical quality attribute.This strategy provides a referential paradigm for palatability enhancement of similar traditional Chinese medicine oral preparations,laying a crucial technical foundation for elevating the clinical value of Chinese herbal medicines and promoting the high-quality development of traditional Chinese medicine(TCM).

Objective To identify the taste critical quality attribute and design and optimize the flavor-correcting formulation of the traditional Chinese medicine oral preparation Qingre Jiedu Oral Liquid,in order to improve its taste and enhance patient medication adherence.Methods The taste assignment method was employed to identify the taste critical quality attribute of Qingre Jiedu Oral Liquid.Based on human sensory evaluation and the standardized Euclidean distance in electronic tongue analysis,suitable types of corrigent were determined.Subsequently,under constraints such as maximum allowable dosage,solubility,and sweetness,the optimal taste formulation for the sugar-free intermediate of Qingre Jiedu Oral Liquid was determined using Box-Behnken experimental design combined with electronic tongue and human sensory evaluation results.The study was reviewed and approved by the Ethics Committee of Beijing University of Chinese Medicine(Ethics Approval Number 2020BZYLL0609).Results The quantitative score for bitter taste of Qingre Jiedu Oral Liquid accounted for 30.36%,confirming bitterness as the taste critical quality attribute requiring attention.The optimal taste formulation for the sugar-free intermediate of Qingre Jiedu Oral Liquid was determined to be 120 mg·mL?1 erythritol,12 mg·mL?1 acesulfame potassium,and 2.4 mg·mL?1 stevioside.This formulation achieved an 11.75-point improvement in sensory evaluation scores compared to the original commercially available oral liquid.Conclusion This study successfully improved the taste of Qingre Jiedu Oral Liquid and established a comprehensive strategy for flavor-correcting formulation optimization,including a method for identifying taste critical quality attribute.This strategy provides a referential paradigm for palatability enhancement of similar traditional Chinese medicine oral preparations,laying a crucial technical foundation for elevating the clinical value of Chinese herbal medicines and promoting the high-quality development of traditional Chinese medicine(TCM).

Objective:To investigate the effects and underlying mechanism of ionizing radiation on the adipogenic of mesenchymal stem cells(MSCs).Methods:Mouse MSCs were cultured in vitro and treated with 2 Gy and 6 Gy radiation with 60Co,and the radiation dose rate was 0.98 Gy/min.Bulk RNA-seq was performed on control and irradiated MSCs.The changes of adipogenic differentiation and oxidative stress pathways of MSC were revealed by bioinformatics analysis.Oil Red O staining was used to detect the adipogenic differentiation ability of MSCs in vitro,and real-time fluorescence quantitative PCR(qPCR)was used to detect the expression differences of key regulatory factors Cebpa,Lpl and Pparg after radiation treatment.At the same time,qPCR and Western blot were used to detect the effect of inhibition of Nrf2,a key factor of antioxidant stress pathway,on the expression of key regulatory factors of adipogenesis.Moreover,the species conservation of the irradiation response of human bone marrow MSCs and mouse MSC was determined by qPCR.Results:Bulk RNA-seq suggested that ionizing radiation promotes adipogenic differentiation of MSCs and up-regulation of oxidative stress-related genes and pathways.The results of Oil Red O staining and qPCR showed that ionizing radiation promoted the adipogenesis of MSCs,with high expression of Cebpa,Lpl and Pparg,as well as oxidative stress-related gene Nrf2.Nrf2 pathway inhibitors could further enhance the adipogenesis of MSCs in bone marrow after radiation.Notably,the similar regulation of oxidative pathways and enhanced adipogenesis post irradiation were observed in human bone marrow MSCs.In addition,irradiation exposure led to up-regulated mRNA expression of interleukin-6 and down-regulated mRNA expression of colony stimulating factor 2 in human bone marrow MSCs.Conclusion:Ionizing radiation promotes adipogenesis of MSCs in mice,and oxidative stress pathway participates in this effect,blocking Nrf2 further promotes the adipogenesis of MSCs.Additionally,irradiation activates oxidative pathways and promotes adipogenic differentiation of human bone marrow MSCs.

Objective:To establish an in vitro cell model simulating acute graft-versus-host disease(aGVHD)bone marrow microenvironment injury with the advantage of mouse serum of aGVHD model and explore the effect of serum of aGVHD mouse on the adipogenic differentiation ability of mesenchymal stem cells(MSCs).Methods:The 6-8-week-old C57BL/6N female mice and BALB/c female mice were used as the donor and recipient mice of the aGVHD model,respectively.Bone marrow transplantation(BMT)mouse model(n=20)was established by being injected with bone marrow cells(1 × 10'per mouse)from donor mice within 4-6 hours after receiving a lethal dose(8.0 Gy,72.76 cGy/min)of y ray general irradiation.A mouse model of aGVHD(n=20)was established by infusing a total of 0.4 ml of a mixture of donor mouse-derived bone marrow cells(1 × 107 per mouse)and spleen lymphocytes(2 × 106 per mouse).The blood was removed from the eyeballs and the mouse serum was aspirated on the 7th day after modeling.Bone marrow-derived MSCs were isolated from 1-week-old C57BL/6N male mice and incubated with 2％,5％and 10％BMT mouse serum and aGVHD mouse serum in the medium,respectively.The effect of serum in the two groups on the in vitro adipogenic differentiation ability of mouse MSCs was detected by Oil Red O staining.The expression levels of related proteins PPARy and CEBPα were detected by Western blot.The expression differences of key adipogenic transcription factors including PPARy,CEBPα,FABP4 and LPL were determined by real-time quantitative PCR(RT-qPCR).Results:An in vitro cell model simulating the damage of bone marrow microenvironment in mice with aGVHD was successfully established.Oil Red O staining showed that the number of orange-red fatty droplets was significantly reduced and the adipogenic differentiation ability of MSC was impaired at aGVHD serum concentration of 10％compared with BMT serum.Western blot experiments showed that adipogenesis-related proteins PPARy and CEBPα expressed in MSCs were down-regulated.Further RT-qPCR assay showed that the production of PPARy,CEBPα,FABP4 and LPL,the key transcription factors for adipogenic differentiation of MSC,were significantly reduced.Conclusion:The adipogenic differentiation capacity of MSCs is inhibited by aGVHD mouse serum.

Objective:To establish a mesenchymal stem cell(MSC)-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease(aGVHD).Methods:Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors,and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients.The recipient mouse received a lethal dose(8.0 Gy,72.76 cGy/min)of total body γ irradiation,and injected with donor mouse derived bone marrow cells(1× 107/mouse)in 6-8 hours post irradiation to establish a bone marrow transplantation(BMT)mouse model(n=20).In addition,the recipient mice received a lethal dose(8.0 Gy,72.76 cGy/min)of total body γ irradiation,and injected with donor mouse derived bone marrow cells(1 × 107/mouse)and spleen lymphocytes(2 × 106/mouse)in 6-8 hours post irradiation to establish a mouse aGVHD model(n=20).On the day 7 after modeling,the recipient mice were anesthetized and the blood was harvested post eyeball enucleation.The serum was collected by centrifugation.Mouse MSCs were isolated and cultured with the addition of 2％,5％,and 10％recipient serum from BMT group or aGVHD group respectively.The colony-forming unit-fibroblast(CFU-F)experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC.The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining.In addition,the expression of self-renewal-related genes including Oct-4,Sox-2,and Nanog in MSC was detected by real-time fluorescence quantitative PCR(RT-qPCR).Results:We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD.CFU-F assay showed that,on day 7 after the culture,compared with the BMT group,MSC colony formation ability of aGVHD serum concentrations groups of 2％and 5％was significantly reduced(P＜0.05);after the culture,at day 14,compared with the BMT group,MSC colony formation ability in different aGVHD serum concentration was significantly reduced(P＜0.05).The immunofluorescence staining showed that,compared with the BMT group,the proportion of MSC surface molecules CD29+and CD 105+cells was significantly dereased in the aGVHD serum concentration group(P＜0.05),the most significant difference was at a serum concentration of 10％(P＜0.001,P＜0.01).The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4,Sox-2,and Nanog was decreased,the most significant difference was observed at an aGVHD serum concentration of 10％(P＜0.01,P＜0.001,P＜0.001).Conclusion:By co-culturing different concentrations of mouse aGVHD serum and mouse MSC,we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability,which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

OBJECTIVE: To investigate the optimal duration of dual antiplatelet therapy (DAPT) in patients with diabetes mellitus (DM) requiring complex percutaneous coronary intervention (PCI). METHODS: A total of 2403 patients with DM who underwent complex PCI from January to December 2013 were consecutively enrolled in this observational cohort study and divided according to DAPT duration into a standard group (11-13 months, n = 689) and two prolonged groups (13-24 months, n = 1133; > 24 months, n = 581). RESULTS: Baseline characteristics, angiographic findings, and complexity of PCI were comparable regardless of DAPT duration. The incidence of major adverse cardiac and cerebrovascular event was lower when DAPT was 13-24 months than when it was 11-13 months or > 24 months (4.6% vs. 8.1% vs. 6.0%, P = 0.008), as was the incidence of all-cause death (1.9% vs. 4.6% vs. 2.2%, P = 0.002) and cardiac death (1.0% vs. 3.0% vs. 1.2%, P = 0.002). After adjustment for confounders, DAPT for 13-24 months was associated with a lower risk of major adverse cardiac and cerebrovascular event [hazard ratio (HR) = 0.544, 95% CI: 0.373-0.795] and all-cause death (HR = 0.605, 95% CI: 0.387-0.944). DAPT for > 24 months was associated with a lower risk of all-cause death (HR = 0.681, 95% CI: 0.493-0.942) and cardiac death (HR = 0.620, 95% CI: 0.403-0.952). The risk of major bleeding was not increased by prolonging DAPT to 13-24 months (HR = 1.356, 95% CI: 0.766-2.401) or > 24 months (HR = 0.967, 95% CI: 0.682-1.371). CONCLUSIONS For patients with DM undergoing complex PCI, prolonging DAPT might improve the long-term prognosis by reducing the risk of adverse ischemic events without increasing the bleeding risk.

Objective: To explore and compare the effect of standard or prolonged dual antiplatelet therapy (DAPT) on the long-term prognosis of elderly patients with coronary heart disease complicated with diabetes mellitus after drug-eluting stent (DES) implantation. Methods: Consecutive patients with diabetes mellitus, ≥65 years old, underwent DES implantation, and had no adverse events within 1 year after operation underwent percutaneous coronary intervention (PCI) from January to December 2013 in Fuwai Hospital were enrolled in this prospective cohort study. These patients were divided into three groups according to DAPT duration: standard DAPT duration group (11 ≤ DAPT duration≤ 13 months) and prolonged DAPT duration group (1324 months). All the patients were followed up at 1, 6 months, 1, 2 and 5 years in order to collect the incidence of major adverse cardiovascular and cerebrovascular events (MACCE), and type 2 to 5 bleeding events defined by the Federation of Bleeding Academic Research (BARC). MACCE were consisted of all cause death, myocardial infarction, target vessel revascularization or stroke. The incidence of clinical adverse events were compared among 3 different DAPT duration groups, and Cox regression model were used to analyze the effect of different DAPT duration on 5-year long-term prognosis. Results: A total of 1 562 patients were enrolled, aged (70.8±4.5) years, with 398 female (25.5%). There were 467 cases in standard DAPT duration group, 684 cases in 1324 months group. The patients in standard DAPT duration group and the prolonged DAPT duration groups accounted for 29.9% (467/1 562) and 70.1% (1 095/1 562), respectively. The 5-year follow-up results showed that the incidence of all-cause death in 13P=0.011) and DAPT duration>24 month group(4.1%(17/411) vs. 8.6%(40/467),P=0.008) were significantly lower than in standard DAPT group. The incidence of myocardial infarction in 13P=0.002). The incidence of MACCE in 1324 month group were 19.3% (90/467), 12.3% (84/684), 20.2% (83/411), respectively, P<0.001). There was no significant difference in the incidence of stroke and bleeding events among the three groups (all P>0.05). Multivariate Cox analysis showed that compared with the standard DAPT group, prolonged DAPT to 13-24 months was negatively correlated with MACCE (HR=0.601, 95%CI 0.446-0.811, P=0.001), all-cause death (HR=0.568, 95%CI 0.357-0.903, P=0.017) and myocardial infarction (HR=0.353, 95%CI 0.179-0.695, P=0.003). DAPT>24 months was negatively correlated with all-cause death (HR=0.687, 95%CI 0.516-0.913, P=0.010) and positively correlated with revascularization (HR=1.404, 95%CI 1.116-1.765, P=0.004). There was no correlation between prolonged DAPT and bleeding events. Conclusions: For elderly patients with coronary heart disease complicated with diabetes mellitus underwent DES implantation, and had no MACCE and bleeding events within 1 year after operation, appropriately prolonging of the DAPT duration is related to the reduction of the risk of cardiovascular adverse events. Patients may benefit the most from the DAPT between 13 to 24 months. In addition, prolonging DAPT duration does not increase the incidence of bleeding events in this patient cohort.
Aged ; Coronary Artery Disease/surgery* ; Diabetes Mellitus ; Drug Therapy, Combination ; Drug-Eluting Stents/adverse effects* ; Female ; Hemorrhage ; Humans ; Male ; Myocardial Infarction/epidemiology* ; Percutaneous Coronary Intervention ; Platelet Aggregation Inhibitors/therapeutic use* ; Prognosis ; Prospective Studies ; Stroke ; Treatment Outcome