[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.

OBJECTIVE To investigate the correlation of serum P-Tau protein and β-amyloid protein expression levels with obstructive sleep apnea syndrome(OSAS)and mild cognitive impairment(MCI)patients,and their diagnostic value.METHODS From December 2020 to December 2023,120 patients with OSAS admitted to Third Hospital of Shijiazhuang were collected as the case group.According to the diagnostic criteria for MCI,patients were grouped into OSAS without MCI group(40 cases)and OSAS with MCI group(80 cases).ELISA method was applied to detect the levels of serum P-Tau protein and β-amyloid protein.Spearman method was applied to analyze the correlation between serum P-Tau protein,β-amyloid protein,and MCI.Multivariate logistic regression was applied to analyze the influencing factors of OSAS patients with MCI.ROC curve was applied to evaluate the diagnostic efficacy of serum P-Tau protein and β-amyloid protein in OSAS patients with MCI.RESULTS The Montreal Cognitive Assessment(MoCA)score in the OSAS with MCI group was obviously lower than that in the OSAS without MCI group(P<0.05).The expression levels of P-Tau protein and β-amyloid protein in the OSAS with MCI group were obviously higher than those in the OSAS without MCI group(P<0.05).The expression levels of serum P-Tau protein and β-amyloid protein in OSAS patients were negatively correlated with MoCA score(r=-0.346,-0.565,P<0.001).Serum P-Tau protein and β-amyloid protein were risk factors for OSAS with MCI(P<0.05).The AUC of the expression levels of serum P-Tau protein,β-amyloid protein,and their combination for OSAS with MCI was 0.751,0.848,and 0.928,respectively.The combined evaluation of the two showed better results(Zcombination-P-Tau protein=4.102,P<0.001;Zcombination-β amyloid protein=2.147,P=0.032).CONCLUSION The expression of serum P-Tau protein and β-amyloid protein is upregulated in OSAS patients with MCI,they are risk factors for the development of MCI in OSAS patients.The combined detection of the two has higher diagnostic efficacy.

Objective To explore the protective effect and possible mechanism of modified Shenqi Dihuang Decoction on podocytes in db/db mice based on ROS/NLRP3/GSDMD signaling pathway.Methods Fifty 8-week-old male db/db mice(SPF grade)were randomly divided into the model group,losartan group and TCM low-,medium-and high-dosage groups,with 10 mice in each group.Ten heterozygous db/m mice served as the blank group.Interventions were administered respectively for 12 weeks.The body mass,random blood glucose,serum creatinine,blood urea nitrogen and 24 h urinary protein content were detected,HE,PAS,PASM,Masson and Sirius red staining was used to observe the morphology of renal tissue,transmission electron microscopy was used to observe the ultrastructure of renal tissue,fluorescent probes were used to observe the release of ROS in renal tissue,immunofluorescence staining was used to detect the expression of Nephrin,NLRP3,Cleaved Caspase-1 and GSDMD-N in renal tissue,and Western blot was used to detect the expression of NLRP3,Cleaved Caspase-1,GSDMD-N,IL-1β,IL-18,Nephrin,Podocin,PODXL,WT-1 and Desmin proteins.Results Compared with the blank group,the body mass and random blood glucose of the model group mice significantly increased(P<0.05),and the contents of serum creatinine,blood urea nitrogen and 24 h urinary protein were significantly increased(P<0.05);glomerular hypertrophy,dilation of renal glomeruli and tubules,thickening of basement membrane,matrix proliferation in mesangial area,abnormal deposition of collagen fibers in renal interstitium,accompanied by damage to renal tubular epithelial structure and focal glomerulosclerosis,significant increase in type Ⅰ collagen deposition,extensive fusion of podocyte processes,and scattered electron dense material in the basement membrane or subepithelial layer;the ROS content in renal tissue significantly increased(P<0.05),and the protein expression of NLRP3,Cleaved Caspase-1,GSDMD-N,IL-1β,IL-18 and Desmin significantly increased(P<0.05),the protein expression of Nephrin,Podocin,PODXL and WT1 significantly decreased(P<0.05).Compared with the model group,the body mass and random blood glucose of mice in each dosage of TCM group were relatively stable,the contents of serum creatinine,blood urea nitrogen and 24 h urinary protein decreased;the pathological damage to renal tissue was reduced,the ultrastructure of podocytes was improved,and the density of podocytes increased;the ROS content decreased,and the protein expression of NLRP3,Cleaved Caspase-1,GSDMD-N,IL-1β,IL-18 and Desmin decreased,while the protein expression of Nephrin,Podocin,WT1 and PODXL increased.With the dosage of modified Shenqi Dihuang Decoction increased,the improvement effect gradually strengthened,and the differences in TCM high-dose group was statistically significant(P<0.05).Conclusion Modified Shenqi Dihuang Decoction can protects podocytes in db/db mice,potentially by modulating the ROS/NLRP3/GSDMD signaling pathway.

Objective To explore the protective effect and possible mechanism of modified Shenqi Dihuang Decoction on podocytes in db/db mice based on ROS/NLRP3/GSDMD signaling pathway.Methods Fifty 8-week-old male db/db mice(SPF grade)were randomly divided into the model group,losartan group and TCM low-,medium-and high-dosage groups,with 10 mice in each group.Ten heterozygous db/m mice served as the blank group.Interventions were administered respectively for 12 weeks.The body mass,random blood glucose,serum creatinine,blood urea nitrogen and 24 h urinary protein content were detected,HE,PAS,PASM,Masson and Sirius red staining was used to observe the morphology of renal tissue,transmission electron microscopy was used to observe the ultrastructure of renal tissue,fluorescent probes were used to observe the release of ROS in renal tissue,immunofluorescence staining was used to detect the expression of Nephrin,NLRP3,Cleaved Caspase-1 and GSDMD-N in renal tissue,and Western blot was used to detect the expression of NLRP3,Cleaved Caspase-1,GSDMD-N,IL-1β,IL-18,Nephrin,Podocin,PODXL,WT-1 and Desmin proteins.Results Compared with the blank group,the body mass and random blood glucose of the model group mice significantly increased(P<0.05),and the contents of serum creatinine,blood urea nitrogen and 24 h urinary protein were significantly increased(P<0.05);glomerular hypertrophy,dilation of renal glomeruli and tubules,thickening of basement membrane,matrix proliferation in mesangial area,abnormal deposition of collagen fibers in renal interstitium,accompanied by damage to renal tubular epithelial structure and focal glomerulosclerosis,significant increase in type Ⅰ collagen deposition,extensive fusion of podocyte processes,and scattered electron dense material in the basement membrane or subepithelial layer;the ROS content in renal tissue significantly increased(P<0.05),and the protein expression of NLRP3,Cleaved Caspase-1,GSDMD-N,IL-1β,IL-18 and Desmin significantly increased(P<0.05),the protein expression of Nephrin,Podocin,PODXL and WT1 significantly decreased(P<0.05).Compared with the model group,the body mass and random blood glucose of mice in each dosage of TCM group were relatively stable,the contents of serum creatinine,blood urea nitrogen and 24 h urinary protein decreased;the pathological damage to renal tissue was reduced,the ultrastructure of podocytes was improved,and the density of podocytes increased;the ROS content decreased,and the protein expression of NLRP3,Cleaved Caspase-1,GSDMD-N,IL-1β,IL-18 and Desmin decreased,while the protein expression of Nephrin,Podocin,WT1 and PODXL increased.With the dosage of modified Shenqi Dihuang Decoction increased,the improvement effect gradually strengthened,and the differences in TCM high-dose group was statistically significant(P<0.05).Conclusion Modified Shenqi Dihuang Decoction can protects podocytes in db/db mice,potentially by modulating the ROS/NLRP3/GSDMD signaling pathway.

Objective: To investigate the effect of caffeine citrate administering at different time on outcome and neurodevelopment of premature infants. Methods: A total of 113 preterm infants with gestational age less than 32 weeks and birth weight less than 1 500 g who were hospitalized and treated in the neonatal intensive care unit from January 2018 to June 2018 were enrolled.According to the time when caffeine citrate treatment was started, they were divided into early treatment group(≤1 days) with 53 infants and late treatment group(1 to 10 days) with 60 infants.A retrospective analysis was performed for their clinical data.The perinatal conditions, treatment process and clinical outcomes of the two groups were compared and the neurological development was followed up at 12 months old. Results: Compared with the late treatment group, the early treatment group had a significantly shorter durations of mechanical ventilation time, oxygen therapy time, hospitalization days and a significantly lower incidence of bronchopulmonary dysplasia, patent ductus arteriosusand intraventricular hemorrhage or periventricular leukomalacia, and there were significant differences between two groups(P<0.05, respectively). The neonatal behavioral neurological assessment score in the early treatment group was higher than that in the late treatment group at 40 weeks of gestational age, and there was significant difference between two groups(P<0.05). The mental developmental index at 3 months of corrected age, the mental developmental index and psychomotor developmental index at 12 months were significantly better in the early treatment group than those in the late treatment group, and there were significant differences between two groups(P<0.05, respectively). Conclusion Early use of caffeine citrate can improve the outcome of premature infants and improve the prognosis of nervous system.

Objective:To examine the prospective association of pubertal timing and tempo with depressive symptoms in adolescents.Methods:Since 2013, 2 084 students in grade 1-3 were selected from two primary schools in Bengbu, Anhui Province were selected by using convenience sampling method to establish the adolescence pubertal development cohort. Followed up for 6 years, physical examination, secondary sexual development evaluation (testicular volume for boys and breast development for girls) and depressive symptoms were evaluated biennially. Non-linear growth model was used to estimate pubertal timing and tempo for boys and girls respectively. Depressive symptoms were interviewed by using the Short Mood & Feeling Questionnaire (SMFQ) at baseline and Mood & Feeling Questionnaire (MFQ) during follow-up for students in grade 1-2. Children Depression Inventory (CDI) was used for students in grade 3 at baseline and during follow-up. Depressive symptom scores were standardized by using the Z-score method. Multivariate linear regression model was used to analyze the predictive effects of modeling pubertal timing and tempo on depressive symptoms of adolescence boys and girls. Results:There were 1 909 students with complete questionnaire and puberty development information, including 1 052 boys (59.19%) and 857 girls (43.81%), with average age about (13.94±0.87) years and 91.60 percent follow-up rate. The average modeling pubertal timing of girls (11.25 years) was earlier than that of boys (12.70 years), and the average pubertal tempo of girls about 1.47 Tanner stage/year was faster than that of boys about 1.28 Tanner stage/year. After controlling for depressive symptoms, maternal education and adverse childhood experiences at baseline and age, body mass index (BMI) classification and sleep time during follow-up, this predictive effect of pubertal timing and tempo on depressive symptoms was only significant among girls. Compared with girls with on time pubertal timing, girls in the delay timing group had a lower level of depressive symptoms (β=-0.19, 95% CI:-0.34,-0.01). Compared with girls in average pubertal tempo group, the fast tempo group associated with an increasing risk of depressive symptoms (β =0.23, 95% CI: 0.05, 0.40), while the slow tempo group associated with an decreasing risk of depressive symptoms (β =-0.21, 95％ CI:-0.39,-0.03). Insignificant effects were found in puberty timing and tempo on depressive symptoms of boys ( P>0.05). Conclusion:Fast pubertal tempo increases the risk of development of depressive symptoms of adolescent girls. There is no predictive effect of pubertal timing and tempo on depression symptoms of adolescent boys.

Objective:To examine the prospective association of pubertal timing and tempo with depressive symptoms in adolescents.Methods:Since 2013, 2 084 students in grade 1-3 were selected from two primary schools in Bengbu, Anhui Province were selected by using convenience sampling method to establish the adolescence pubertal development cohort. Followed up for 6 years, physical examination, secondary sexual development evaluation (testicular volume for boys and breast development for girls) and depressive symptoms were evaluated biennially. Non-linear growth model was used to estimate pubertal timing and tempo for boys and girls respectively. Depressive symptoms were interviewed by using the Short Mood & Feeling Questionnaire (SMFQ) at baseline and Mood & Feeling Questionnaire (MFQ) during follow-up for students in grade 1-2. Children Depression Inventory (CDI) was used for students in grade 3 at baseline and during follow-up. Depressive symptom scores were standardized by using the Z-score method. Multivariate linear regression model was used to analyze the predictive effects of modeling pubertal timing and tempo on depressive symptoms of adolescence boys and girls. Results:There were 1 909 students with complete questionnaire and puberty development information, including 1 052 boys (59.19%) and 857 girls (43.81%), with average age about (13.94±0.87) years and 91.60 percent follow-up rate. The average modeling pubertal timing of girls (11.25 years) was earlier than that of boys (12.70 years), and the average pubertal tempo of girls about 1.47 Tanner stage/year was faster than that of boys about 1.28 Tanner stage/year. After controlling for depressive symptoms, maternal education and adverse childhood experiences at baseline and age, body mass index (BMI) classification and sleep time during follow-up, this predictive effect of pubertal timing and tempo on depressive symptoms was only significant among girls. Compared with girls with on time pubertal timing, girls in the delay timing group had a lower level of depressive symptoms (β=-0.19, 95% CI:-0.34,-0.01). Compared with girls in average pubertal tempo group, the fast tempo group associated with an increasing risk of depressive symptoms (β =0.23, 95% CI: 0.05, 0.40), while the slow tempo group associated with an decreasing risk of depressive symptoms (β =-0.21, 95％ CI:-0.39,-0.03). Insignificant effects were found in puberty timing and tempo on depressive symptoms of boys ( P>0.05). Conclusion:Fast pubertal tempo increases the risk of development of depressive symptoms of adolescent girls. There is no predictive effect of pubertal timing and tempo on depression symptoms of adolescent boys.

Objective: To investigate the longitudinal association between peer bullying victimization and internalizing symptoms, and to examine whether parental warmth buffers against this effect,so as to provide a reference for improving the mental health of the special group of children. Methods: Students in grade 4 and grade 5 from three primary schools in Ma’anshan, Anhui Province were selected in Oct. 2017 with informed consent. Height, weight and pubertal development were objectively assessed. Internalizing symptoms and peer bullying victimization were evaluated at baseline and 1-year follow-up by using the MacArthur Health and Behavior Questionnaire-Child. Children were classified into three groups as non-bullying, non-consistent and consistent bullying victimization group. Multiple linear regression model was used to analyze whether the association between peer bullying victimization and internalizing symptoms varied across different parental warmth groups. Results: Compared with non-bullying victimization group, consistent-and non-consistent bullying victimization had higher internalizing symptoms at baseline and 1-year follow-up(P<0.01). Students reported consistent, non-consistent bullying victimization under moderate parental warmth had lower internalizing symptoms at baseline and 1-year follow-up than those reported under low parental warmth group(P<0.01). Multiple liner regression showed that consistent bullying victimization [β=0.66(95%CI=0.33-0.99), 0.37(95%CI=0.15-0.59), 0.58(95%CI=0.31-0.84), P<0.05] and non-consistent bullying victimization[β=0.33(95%CI=0.18-0.47), 0.28(95%CI=0.13-0.41), 0.29(95%CI=0.08-0.51),P<0.05] were associated with higher 1-year follow-up internalizing symptoms in non-consistent, moderate-and low parental warmth group, while such association was not observed in consistent parental warmth group. Abstract The experience of peer bullying victimization increases the risk of internalizing symptoms and this finding suggests that parental warmth help to buffer children from the internalizing symptoms associated with bullying victimization.

Objective: To examine the prospective effect of cesarean section on pubertal development after controlling the polygenic susceptibility for early puberty in boys and girls,and to provide a reference for choosing a delivervary way beneficial to pubertal development. Methods: Cluster sampling method was used to select 997 students of grade 1 to 3 from 2 priwary schools in Bengbu of Anhui in 2016 to establish the puberty development cohort. Delivery mode,birth weight,gestational age,early teeding way,family income and parental educational background were derived from parental questionnaire. Breast Tanner stage and testicular volume of 997 children were annually assessed. The polygenic risk score (PRS) was computed based on 17 SNPs derived from published genome-wide association studies for early pubertal timing. Kaplan-Meier analyses was used to examine associations between cesarean section with pubertal development in boys and girls. Results: The average age of 997 children was 8.01±0.84 years old, and average BMI was (17.96±2.96) kg/m2. The prevalence of cesarean section for boys and girls was 43.30% and 32.47%, respectively. At baseline, the detection of thelarche in girls delivered by cesarean section (39.89%) was higher than that of girls delivered naturally (4.39%) (χ2=118.65, P<0.01). Similar result was observed during the last follow-up, the detection of thelarche in girls born by cesarean section (95.21%) was higher than girls born vaginally (68.48%), respectively (χ2=51.42, P<0.01). After adjusting for confounding factors (age, BMI, birthweight, infancy feeding, gestational age, household monthly income and parental education), girls delivered by cesarean section had maturation at significantly younger than girls who delivered naturally (Hazard Ratio, HR=1.98，95%CI=1.67-2.34，P<0.01). Conclusion Caesarean section might increase the risk of earlier age at pubertal onset in a sex-specific manner. Whether natural delivery could be one of the protective measures to prevent early pubertal timing in children warrants further investigation.

Objective: To explore the impact of adverse childhood experiences on pubertal development,and to provide a reference for conducting the targeted intervention in the early stage. Methods: A cluster sampling method was used to randomly select students of grades 3 to 4 from three primary schools in Ma’anshan municipality of Anhui province in October 2017. Self-reported childhood adverse experiences (ACEs), including physical, emotional and sexual abuse, as well as physical and emotional neglect were collected. Testicular volume and breast Tanner stage were assessed. A follow-up survey was conducted one year later to assess ACEs and pubertal development. ACEs of each dimension were divided into non-exposure group, transient exposure group and sustained exposure group according to ACEs exposure at baseline and follow-up. Multivariate linear regression model was used to analyze the relationship between ACEs exposure and pubertal development after 1-year follow-up. Results: Sustained emotional abuse was positively associated with increased breast Tanner stage and testicular volume (breast: β=0.36, 95%CI=0.09-0.63, P<0.01; testicular volume: β=1.07, 95%CI=0.47-1.66, P<0.01); sustained physical abuse was positively associated with decreased testicular volume (β=-0.83, 95%CI=-1.58--0.08, P<0.05); sustained and transient sexual abuse were significant positively associated with decreased breast Tanner stages (sustained: β=-1.43, 95%CI=-2.86--0.02, P<0.05; transient: β=-0.45, 95%CI=-0.73--0.19, P<0.05); sustained emotional neglect was positively associated with increased breast Tanner staging (β=0.33, 95%CI=0.06-0.61, P<0.05) and decreased testicular volume (β=-0.19, 95%CI=-1.38--0.19, P<0.01). Conclusion Sustained emotional abuse was associated with early pubertal development in boys and girls,and the specitic mechanisam differences of different ACES puberty development effects and the gender differences of the same ACES puberty development effects need to be furthur explored.