[Objective] To explore the feasibility of using whole blood as a source of NK cells for allogeneic CAR NK cell therapy and activated NK cell reinfusion therapy, and initially construct a technical system for the separation and purification of NK cells from whole blood. [Methods] All peripheral blood mononuclear cells (PBMCs) were enriched from 400 mL of whole blood by manual separation and machine separation, respectively. The erythrocyte loss rate, PBMCs number, NK cell purity of the two methods were compared. NK cells were sorted from PBMCs by three separation and enrichment methods as immunomagnetic bead negative selection method, platelet lysate culture expansion and PERCOLL density gradient separation method, and the purity and yield of NK cells, the activity of NK cells and the tumor-killing ability of the three separation and enrichment methods were compared. [Results] The proportion of NK cells in the lymphocyte population was higher in the manual separation method than in the machine separation method[(13.16±5.16)% vs (8.56±3.92)%, P<0.05]; the number PBMCs was lower in the manual separation method than in the machine separation method[(4.09±1.80)×10⁸vs (6.49±2.16)×10⁸, P<0.05], and there was no difference in the red blood cell loss between the two methods (P>0.05). The purity of NK cells isolated and enriched from PBMCs by manual separation method using immunomagnetic was (96.77±2.31)%; the yield was (56.27±10.47)%; the inhibition of tumor proliferation was (38.67±14.05)%; and the tumor killing rate was (19.90±8.05)%. The purity of NK cells isolated and enriched from PBMCs by manual separation method using platelet lysis culture expansion method was the highest at day 7, which was (54.84±15.80)%; the cell expansion multiple could reach 16.92±6.28 at day 7; the in vitro tumor killing rate of NK cells was (15.83±5.5)%; the tumor inhibition rate was (44.33±13.5)%; and there was no difference in the toxicity and activity of NK cells between the two methods (P>0.05). The purity of NK cells isolated and enriched by PERCOLL density gradient separation method was (15.83±5.82)%, and the yield was (14±6.25)%, which was significantly lower than the other two methods. [Conclusion] PBMCs isolated from whole blood by manual separation and NK cells enriched by negative selection with immunomagnetic beads have the potential to provide NK cell materials for CAR-NK cell therapy, and NK cells enriched by platelet lysate-conditioned medium have the potential to provide NK cells for large-scale NK cell activation reinfusion therapy.

ObjectiveTo investigate the triglyceride-glucose (TyG) index and the level of serum homocysteine (Hcy) in association with the incidence of stroke in type 2 diabetes mellitus (T2DM) patients. MethodsBased on the chronic disease risk factor surveillance cohort in Pudong New Area, Shanghai, excluding those with stroke in baseline survey, T2DM patients who joined the cohort from January 2016 to October 2020 were selected as the research subjects. During the follow-up period, a total of 318 new-onset ischemic stroke patients were selected as the case group, and a total of 318 individuals matched by gender without stroke were selected as the control group. The Cox proportional hazards regression model was used to adjust for confounding factors and explore the serum TyG index and the Hcy biochemical indicator in association with the risk of stroke. ResultsThe Cox proportional hazards regression results showed that after adjusting for confounding factors, the risk of stroke in T2DM patients with 10 μmol·L⁻¹-¹ and Hcy>15 μmol·L⁻¹ was 1.532 times (95%CI: 1.017‒2.309 times, P=0.041) and 1.738 times (95%CI: 1.119‒2.699 times, P=0.014) higher respectively, compared to T2DM patients with Hcy≤10 μmol·L⁻¹. T2DM patients with TyG>9.074 had 1.396 times higher risk of stroke (95%CI: 1.091‒1.787, P=0.008) compared to those with TyG≤9.074. The study found that urea and α1-antitrypsin (α1-AT) were independent risk factors for the incidence of stroke in T2DM patients. For every unit increase in urea andα1-AT, the risk of stroke in T2DM patients increased by 233.6% (HR=3.336, 95%CI: 1.588‒7.009, P=0.001) and 11.3% (HR=1.113, 95%CI: 1.028‒1.205, P=0.008), respectively. Cumulative risk curves showed that Hcy>10 μmol·L⁻¹ and TyG>9.074 accelerated the time to stroke occurrence in T2DM patients. ConclusionElevated levels of Hcy>10 μmol·L⁻¹ and a higher TyG index are risk factors for stroke in T2DM patients. Effective interventions for Hcy and TyG should be conducted for diabetic patients to reduce the incidence of stroke.

ObjectiveTo investigate the correlation between iron metabolism parameters and various syndrome types of unstable angina pectoris （UAP）. MethodsA cross-sectional study was conducted from October 2021 to October 2023， encompassing 213 patients diagnosed with UAP at Xiyuan Hospital of Chinese Academy of Chinese Medical Sciences. Additionally， 30 healthy individuals were selected as control cases. Single-factor analysis was used to investigate the differences in clinical data among different Traditional Chinese Medicine （TCM） syndrome types of UAP and their correlation with iron metabolism indices. The study conducted a comparative analysis of the aforementioned clinical data among patients with and without heat-toxic and blood-stasis syndrome. Logistic regression was used to analyze the correlation between TCM syndrome types and related factors. The receiver operator characteristic （ROC） curve was employed to assess the predictive value of iron metabolism indices， along with their sensitivity and specificity. ResultsCompared to those in the control group， serum iron （SI） and serum ferritin （SF） levels were significantly increased in the UAP group （P<0.01）， while transferrin （TRF） and total iron binding capacity （TIBC） levels were decreased （P<0.01）. However， there was no significant difference in unsaturated iron binding capacity （UIBC）. Multivariate binary Logistic regression analysis identified apolipoprotein A1 （ApoA1）， homocysteine （HCY）， high-sensitivity C-reactive protein （hs-CRP）， and SF as independent influencing factors for the UAP patients （P<0.05， P<0.01）. Additionally， statistically significant differences were observed in SI， SF， TRF， and TIBC among 213 patients with different TCM types （P<0.01）. Patients with heat-toxic and blood-stasis syndrome had higher SI and SF values than those without the syndrome （P<0.01）， while their TIBC and TRF values were lower （P<0.01）. Multivariate binary logistic regression analysis showed that SI and LDL-C levels were closely associated with the differentiation of heat-toxic and blood-stasis syndrome. ConclusionUAP patients often experience iron metabolism disorders， and the heat-toxic and blood-stasis syndrome are significantly correlated with iron metabolism parameters. The SI and LDL-C levels have high specificity and sensitivity in diagnosing heat-toxic and blood-stasis syndrome.

Objective Zinc finger protein 335 (Zfp335) plays a crucial role in the early development of thymic T cells and the differentiation of peripheral T cell subpopulations. The objective of this study is to investigate the role and underlying mechanisms of Zfp335 in the regulation of regulatory T cell (Treg) within tumor immunity. Methods The Zfp335 gene was specifically knocked out in Treg using tamoxifen (Zfp335^fl/fl FOXP3^creERT2), and the MC38 tumor model was established. On the 7th day after tumor inoculation, tumor size was observed and measured. Tumor size was monitored and recorded daily starting from day 7 post-inoculation. On day 12, tumors were harvested, and the proportions of CD4⁺ T cells, CD8⁺ T cells, and Treg were analyzed by flow cytometry. Additionally, the mitochondrial function of effector regulatory T cell (eTreg) was assessed. Results From day 10 post-tumor inoculation, tumor volume in the Zfp335^CKO group was significantly reduced compared to that of the wild-type (WT) group. Furthermore, the infiltration of CD4⁺ and CD8⁺ T cells, along with their respective effector cells, was significantly higher in the Zfp335^CKO group than in the WT group. The proportions of CD4⁺ and CD8⁺ T cells producing interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) were also significantly increased in the Zfp335^CKO group compared to that of the WT group. In addition, the percentage of CD8⁺ T cells secreting granzyme B (GzmB) was significantly higher in the Zfp335^CKO group than that in the WT group. In contrast, the proportion of Treg and inducible T cell co-stimulator (ICOS)⁺ Treg in the Zfp335^CKO group was significantly lower than that in the WT group. Finally, the expression level of Mitotracker Deep Red in eTreg from the Zfp335^CKO group was significantly reduced compared to that in the WT group. Conclusion During tumorigenesis, the specific deletion of Zfp335 impairs Treg activation, which is related to decreased mitochondrial function in eTreg. In Zfp335^CKO mice. Tumors exhibit increased infiltration of effector T cells, accompanied by elevated levels of cytotoxic cytokines, ultimately enhancing resistance to tumor progression.
Animals ; T-Lymphocytes, Regulatory/metabolism* ; Mice ; CD8-Positive T-Lymphocytes/immunology* ; Neoplasms/genetics* ; Cell Line, Tumor ; Mice, Inbred C57BL ; Mice, Knockout ; DNA-Binding Proteins/genetics* ; Female

Objective:To explore the clinical characteristics, audiological outcomes, and factors influencing the efficacy of pharmacological treatment in patients with sudden hearing loss associated with large vestibular aqueduct syndrome（LVAS）. Methods:A retrospective analysis was conducted on the clinical data of 77 bilateral LVAS patients（117 ears） hospitalized for sudden hearing loss from January 1, 2009, to December 31, 2023. The inclusion criteria required that patients to be diagnosed according to the Valvassori standard and had received standardized pharmacological treatment. Clinical features, audiological outcomes, and treatment efficacy were analyzed. Statistical methods were employed to identify factors associated with treatment outcomes. Results:The age of the enrolled patients ranged from 4 to 37 years. The age of onset for the initial hearing fluctuation varied between 0 and 24 years, with a mean age of 5.8 years. The male-to-female ratio was approximately balanced（37 males and 40 females）. The proportion of unilateral to bilateral sudden hearing loss was 1.0︰1.2, with unilateral right ear hearing loss being more frequently occurring（64.9%）. Triggering Factors: Triggers included no identifiable factors in 48.1% of cases, a history of head trauma（24.7%）, upper respiratory tract infections（11.7%）, onset following physical fatigue（11.7%）, and less frequently, noise exposure, alcohol consumption, or emotional stress（each 1.3%）. Clinical Symptoms: Hearing loss was the sole symptom in 35.1% of cases. Concurrent symptoms included vertigo in 44.2% and tinnitus in 46.8%. Patients with a disease duration of ≤14 days demonstrated a treatment efficacy rate of 75.0%. Among those who responded to treatment, 93.0% had profound or greater hearing loss prior to therapy, with an average improvement in hearing thresholds of 32 dB HL. In pretreatment, 68.9% of patients exhibited low-frequency air-bone gaps, increasing to 76.1% post-treatment. Additionally, 17.6% of treated ears demonstrated a ≥15 dB HL improvement in low-frequency bone conduction thresholds. In the non-responsive group, 7.3% of ears still showed some improvement in bone conduction thresholds. Statistically significant differences（P<0.05） were observed between the treatment-effective and non-effective groups concerning the age of initial hearing fluctuation, disease duration, and severity of hearing loss at onset. Conclusion:The efficacy of pharmacological treatment for sudden hearing loss in LVAS patients is influenced by the age at onset, duration of the disease, and severity of hearing impairment. Early diagnosis and timely intervention significantly enhance treatment efficacy, particularly in patients with a disease duration of ≤14 days and an initial sudden hearing loss. Patients with severe hearing loss, especially those with profound or greater impairment, exhibit greater sensitivity to treatment. Pharmacological interventions positively impact both air conduction and bone conduction thresholds, with the observed improvement in bone conduction thresholds warranting further investigation.
Humans ; Male ; Retrospective Studies ; Hearing Loss, Sudden/etiology* ; Female ; Vestibular Aqueduct/pathology* ; Adult ; Child ; Adolescent ; Child, Preschool ; Young Adult ; Treatment Outcome

Objective To investigate the clinical application value of the non-shared incentive diffusion weighted imaging(ZOOM-DWI)technique at cervical level in diagnosis of cervical spondylotic myelopathy(CSM).Methods A total of 49 CSM patients(patient group),and 50 healthy volunteers(control group)were recruited.All subjects underwent conventional MRI and ZOOM-DWI of the cervical spine and neurologic modified Japanese Orthopaedic Association(mJOA)scores in patients with CSM.The apparent diffusion coefficient(ADC)value in the spinal cord at the narrowest area(C5-C6)of the compression site of patients,the ADC value at the disc level in each upper and lower level,and the spinal ADC value at the cervical level C2-C3 were measured.The ADC values of control group C2-C3,C3-C4,C4-C5,C5-C6,C6-C7 were measured.Within-group comparisons of the spinal cord ADC values for each segment between patient and control groups were performed using analysis of variance and post hoc comparisons(SNK-q).The ADC values at the narrowest point of the patient group and control group were tested by independent sample t-test.The Pearson correlation analysis was performed between patients'C5-C6 ADC values and mJOA scores.Results The mean ADC values showed no significantly different levels in the control group.Among the ADC values at each measurement level in the patient group,except for C4-C5 and C6-C7 where the difference was not statistically significant,the remaining pair-wise comparisons all showed statisti-cally significant differences(F=24.368,P＜0.001),with the highest ADC value at C5-C6.The C5-C6 ADC value in the patient group was significantly higher compared to the control group(t=9.414,P＜0.001),with statistical significance.The ADC values at the patient stenosis showed a significant negative correlation with the mJOA score(r=-0.493,P＜0.001).Conclusion Cervical ZOOM-DWI technique can be applied to diagnose CSM,and spinal ADC values can be used as reliable imaging data for diagnosing CSM.

[Objective]To discuss the occurrence and gastric cancer accompanied by depression from the perspective of"depression caused by illness"and"illness caused by depression",and provide theoretical support for the treatment by traditional Chinese medicine(TCM).[Methods]By consulting the ancient and modern literature of TCM,and collecting the academic views of some doctors on the theory of"depression caused by illness"and"illness caused by depression"of gastric cancer,combined with clinical practice experience,it discusses the diagnosis and treatment of gastric cancer accompanied by depression.[Results]The etiology and pathogenesis of gastric cancer accompanied by depression can be summarized as follows:loss of middle Qi,the rise and fall of surly for its root;cementation of stasis and toxin,seven emotions disorder is its root;tumor gathering locally,poison overflowing into the blood vessels,illness and depression intermingled as its symptom,and it accordingly points out that the treatment of gastric cancer accompanied by depression should mediate middle Qi and smooth the Qi transformation of spleen and stomach;regulate Qi and open depression,regulate the rise and fall of Qi of the body;remove blood stasis and detoxify,dredge the body's Qi,blood and body fluid;form and spirit in tune,restore the complex of the five viscera of the human body.[Conclusion]Stasis,toxin and depression are important pathological factors affecting the occurrence,development and outcome of gastric cancer accompanied by depression.These three factors often affect each other as causation.Discussion of gastric cancer accompanied by depression from the perspective of"depression caused by illness"and"illness caused by depression"is not only helpful to improve the etiological and pathogenesis theory of gastric cancer accompanied by depression,but also helpful to provide theoretical reference for its clinical treatment.

Objective To screen the active components of total flavonoid extracts of Sarcandra glabra to promote megakaryocyte differentiation.Methods(1)A model of megakaryocyte differentiation disorder was established by co-culturing human megakaryocytic leukaemia cells(Dami)with human bone marrow stromal cells(HS-5)as an evaluation system,and the experimental groupings were as follows:the Dami group(Dami),the control group(Dami+HS-5),and the PMA group[Dami+HS-5+5 ng·mL-1 foprolol 12-tetradecanoate 13-acetate(PMA)],and model group[Dami+HS-5+1%rabbit anti-rat platelet serum(APS)+5 ng·mL-1 PMA]were cultured for 48 hours.The expressions of megakaryocyte differentiation and maturation surface marker molecules,CD41a and CD61 were detected by flow cytometry.(2)Forty-nine SD male rats were randomly divided into blank plasma group,15-minute group,30-minute group,60-minute group,90-minute group,120-minute group,and 240-minute group,with 7 rats in each group.The rats in each administration group were gavaged with 1.26 g·kg-1 of total flavonoids extracts of Sarcandra glabra,and blood was collected at six set time points(15,30,60,90,120,240 minutes)for the preparation of time-dependent serum-containing plasma of total flavonoids extracts of Sarcandra glabra.(3)Ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry(UHPLC-Q-TOF/MS)was used to analyze the plasma of the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra,and the peak area was used to construct a matrix(X-matrix)of the amount of chemical composition change over time in the time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra.The collected time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra at six different time points was used to intervene in the model of megakaryocyte differentiation and maturation disorder,and the expression of cell surface molecules CD41a and CD61 was detected by flow cytometry to construct the matrix of effect of time-dependent serum-containing plasma of the total flavonoids extracts of Sarcandra glabra(Y-matrix).(4)After the data of X and Y matrices were standardized,partial least squares(PLS)was used to calculate and analyze the quantitative and qualitative effect relationship,and variable importance for projection(VIP)>1 was used as the threshold to screen the effect components related to the changes of cell surface molecules CD41a and CD61,and chemical composition identification,as the potential effector components in the total flavonoid extracts of Sarcandra glabra were used to promote the differentiation of megakaryocytes,and finally the regression evaluation system was used to verify the efficacy of its medicinal effect.Results(1)Compared with the Dami group,the expression level of CD41a on the surface of Dami cells in the control group was significantly increased(P＜0.05).Compared with the control group,the expression levels of CD41a and CD61 on the surface of Dami cells in the PMA group were significantly increased(P＜0.01).Compared with the PMA group,the expression levels of CD41a and CD61 on the surface of Dami cells in the model group were significantly reduced(P＜0.01).(2)Compared with the blank plasma group,the expression levels of the molecules CD41a and CD61 on the surface of Dami cells at each time point of 15,30,60,90,120,and 240 minutes were significantly increased(P＜0.01),and the expression levels of CD41a and CD61 were both highest in the 30-minute group.The potential effective components with VIP value greater than 1 were screened out in the positive and negative ion mode,and 540.3638@12.25 and 559.2991@11.53 were selected for pharmacodynamic verification.559.2991@11.53 was identified as daucosterol(Dau),540.3638@12.25 was identified as rosmarinic acid 4-O-β-D-glucoside(Ros).After Ros and Dau intervened in the megakaryocyte differentiation and maturation disorder model respectively,the expression levels of CD41a and CD61 on the surface of Dami cells in the low-,medium-and high-dose groups(40,60 and 80 μg·mL-1)of Ros and Dau were significantly increased compared with the model group(P＜0.05,P＜0.01).Conclusion Ros and Dau may be the active components of the total flavonoids extracts of Sarcandra glabra to promote the differentiation of megakaryocytes.

With the high-speed growing demands for biomedical materials, graphene oxide(GO) and its functional derivatives show broad application prospects in the system of drug delivery, tumor therapy and nanobiology. GO has excellent hydrophilicity and easily modified surface properties, due to a large content of oxygen-containing functional groups on the GO surface. Meanwhile, the huge specific surface areas and unique π-electronic structures give GO adhesion and adsorption capacities. By means of π-π stacking, electrostatic interaction, hydrophobic interaction or covalent bonding, therapeutic agents can be effectively combined with GO to construct drug delivery systems. GO plays an important role in transdermal drug delivery systems due to their biocompatibility, antibacterial activity, and pro-wound healing activity. As a novel drug delivery system, GO nanomaterials can achieve targeted drug delivery, accumulate drug concentrations at the focal site, and improve drug delivery efficiency. Utilizing the promoting skin regeneration and wound healing properties of GO, several antibacterial materials and wound dressings are prepared for wound treatment. In addition, the special far-infrared resonance effect and electrothermal effect of GO result in heat and electric transfer. In other words, graphene oxide could control and promote transdermal absorption efficiency upon the stimuli of heat and electric. GO is expected to break through the limitation of low transdermal absorption rates in the transdermal drug delivery systems and achieve the purpose of enhancing the transdermal permeability of target molecules (especially macromolecules): ultimately improving bioavailability in vivo. This paper will introduce the structures, physicochemical properties, and biological activities of graphene oxide, summarize its application in transdermal drug delivery systems such as drug carriers, antibacterial materials, wound dressings and transdermal enhancers, and prospect its application in clinical medicine. We hope to provide new ideas and different point of views for the biomedical research of graphene oxide and derivatives. GO will become a representative biomaterial in the field of precision medicine and play a key role in the diagnosis and treatment of diseases.