Objective To identify the independent predictors of programmed death-1 (PD-1) inhibitor-related endocrine adverse events and construct a clinically usable risk prediction model. Methods A total of 302 patients with solid tumors treated with PD-1 inhibitors were retrospectively enrolled. According to the presence or absence of endocrine immune-related adverse events (irAEs), the patients were divided into case group and control group. The clinical and laboratory indexes were compared between the two groups. Multivariable logistic regression was used to confirm independent predictors of endocrine irAEs. The nomogram was constructed, while the receiver operating characteristic (ROC) curve was used to test the prediction performance of the model. Results The overall incidence of endocrine irAEs was 21.9% (66/302), and the incidence of hypothyroidism was 19.5% (59/302). The age, PD-1 inhibitors, free thyroxine, thyroid peroxidase antibody (TPOAb), thyroglobulin, amylase, lymphocyte subset CD3 expression were statistically different between the two groups (P＜0.05). Multivariable logistic regression showed that higher expression of lymphocyte subset CD3 was a protective factor to prevent endocrine irAEs occurrence (P＝0.004), while age＜60 years, higher TPOAb and use of pembrolizumab were independent risk factors of endocrine irAEs (P＜0.05). The nomogram model thus constructed, and when the threshold probability of the model exceeded 0.1, its net benefit was higher. ROC curve showed that the AUC of the model to predict endocrine irAEs was 0.760. The prediction result of the model was highly consistent with the actual result. Conclusions The age, type of PD-1 inhibitor, baseline TPOAb level, and baseline CD3 expression can independently predict endocrine irAEs occurrence or not. The nomogram model based on this model has good predictive efficiency, which can provide reference for early identification of high-risk patients and immunotherapy management.

Objective:To investigate damaging effects of clomifene citrate(CC)on endometrial stromal cells(hEndoSCs),and to study effects of spermidine on autophagy and inflammatory cytokine expression in damaged endometrial stromal cells.Methods:Groups were firstly divided into control group,spermidine group,clomiphene group(CC group),CC+Spermidine group.MTT assay was used to detect cell survival rate of hEndoSCs after co-incubation with different concentrations of CC or Spermidine for 24 h.Con-tent of intracellular reactive oxygen species(ROS)and level of apoptosis in cells of the 4 groups were detected by flow cytometry tech-nique.Western blot was used to detect expressions of autophagy pathway-related proteins ULK1,p-ULK1,LC-3Ⅱ,and apoptosis-re-lated proteins Bax,Bcl-2,Cleaved-caspase 3.RT-qPCR was used to detect mRNA expressions of IL-6,IL-1β and TNF-α.Results:Compared with control group,CC group showed decreased cell survival,increased apoptosis rate,ROS content,Bax,Cleaved-cas-pase 3 expressions,decreased Bcl-2 expression,decreased levels of autophagy-related proteins p-ULK1 and LC-3Ⅱ/Ⅰ,and elevated expressions of inflammatory factors IL-6,IL-1β and TNF-α mRNA(P<0.01).There was no significant changes viability of cells in spermidine group compared with control group(P>0.05).Compared with CC group,cell survival rate in CC+spermidine group was sig-nificantly increased,apoptosis rate,ROS content,Bax and Cleaved-caspase 3 expressions were decreased,Bcl-2 expression was in-creased,expressions of autophagy-related proteins p-ULK1 and LC-3Ⅱ/Ⅰ were elevated,while expressions of inflammatory factors IL-6,IL-1β and TNF-α mRNA were decreased(P<0.01).Conclusion:CC can inhibit endometrial stromal cell proliferation,promote apoptosis,and increase the transcript levels of inflammatory factors IL-6,IL-1β and TNF-α.Spermidine can reduce intracellular ROS in clomiphene-injured endometrial stromal cells by activating cellular autophagy,increase cell survival,and inhibit the expressions of inflammatory factors IL-6,IL-1β and TNF-α.

Seizures are a prevalent clinical manifestation during the neonatal period.The majority of neonatal seizures are transient symptoms caused by acute lesions，but in a few cases they are developmental epileptic encephalopathy with neonatal onset.Continuous video-EEG monitoring is the gold standard for diagnosing seizures in newborns,and complex seizures require comprehensive judgment using multi-channel monitoring such as synchronized EMG,blood oxygen,ECG,and respiratory monitoring.In 2021,the International League Against Epilepsy (ILAE) published the updated guidelines for the classification of neonatal seizures,proposing that neonatal seizures cannot be diagnosed if there is only clinical seizure without EEG evidence.Instead,they should be categorized into two types based on the presence of clinical movements and the characteristics of EEG changes during the seizure: electroclinical seizures and electrical seizures.Electroclinical seizures are categorized into motor seizures,non-motor seizures,sequential seizures,and unclassified seizures based on the symptoms exhibited during an attack.In 2023,the ILAE developed evidence-based recommendations for the management of antiseizure medications,discussed the treatment of neonatal seizures based on the latest evidence and emphasized the importance of video EEG in the treatment of neonatal seizures.Phenobarbital remains the first-line antiseizure medication,with phenytoin,levetiracetam,midazolam,or lidocaine as second-line agents.Pyridoxine should be considered for the treatment of neonatal seizures when first- and second-line drugs are ineffective.

Objective:To discuss the effect of human umbilical cord mesenchymal stem cells culture supernatant(hUCMSCs-Sup)on the proliferation,apoptosis,and endometrium receptivity of the human endometrial stromal cells(hEndoSCs)treated with mifepristone(Ms),and to clarify the possible mechanism.Methods:The hEndoSCs were cultured in vitro and divided into control group and 40,60,80,and 100 μmol·L-1 Ms groups.The survival rates of the cells in various groups were detected by MTT assay.The hEndoSCs were divided into control group,40 μmol·L-1 Ms group,and 60 μmol·L-1 Ms group.The apoptotic rates of the cells in various groups were detected by flow cytometry;the expression levels of apoptosis-related protein B-cell lymphoma-2(Bcl-2)and Bcl-2-associated X protein(Bax)proteins in the cells in various groups were detected by Western blotting method,and the ratio of Bcl-2/Bax was calculated.After treated with hUCMSCs-Sup,the hEndoSCs were divided into control group,Ms group,Ms+hUCMSCs-Sup group,and Ms+hUCMSCs-Sup+3-methyladenine(3-MA)group.The survival rates of the cells in various groups were detected by MTT assay;the apoptotic rates of the cells in various groups were detected by flow cytometry;the expression levels of microtubule-associated protein 1 light chain 3B-Ⅱ(LC3B-Ⅱ)and microtubule-associated protein 1 light chain 3B-I(LC3B-Ⅰ)proteins in the cells in various groups were detected by Western blotting method,and the ratio of LC3B-Ⅱ/LC3B-Ⅰwas calculated;the expression levels of endometrium receptivity marker molecules mRNA in the cells in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method.Results:Compared with control group,the survival rates of the cells in 40,60,80,and 100 μmol·L-1 Ms groups were significantly decreased(P<0.05)in a time-dependent and dose-dependent manner.Compared with control group,the apoptotic rates of the cells in 40 and 60 μmol·L-1 Ms groups were significantly increased(P<0.05),and the ratios of Bcl-2/Bax were significantly decreased(P<0.05).After treated with hUCMSCs-Sup,compared with control group,the survival rate of the cells and ratio of LC3B-Ⅱ/LC3B-Ⅰ in the cells in Ms group were significantly decreased(P<0.05),the apoptotic rate was significantly increased(P<0.05),and the expression levels of homeobox A10(HOXA10),leukemia inhibitory factor(LIF),and integrin subunit beta 3(ITGB3)mRNA in the cells were significantly decreased(P<0.05);compared with Ms group,the survival rate of the cells and ratio of LC3B-Ⅱ/LC3B-Ⅰin the cells in Ms+hUCMSCs-Sup group were significantly increased(P<0.05),the apoptotic rate was significantly decreased(P<0.05),and the expression levels of HOXA10,LIF,and ITGB3 mRNA in the cells were significantly increased(P<0.05);compared with Ms+hUCMSCs-Sup group,the survival rate of the cells and ratio of LC3B-Ⅱ/LC3B-Ⅰ in the cells in Ms+hUCMSCs-Sup+3-MA group were significantly decreased(P<0.05).Conclusion:hUCMSCs-Sup can increase the survival rate and decrease the apoptotic rate of the hEndoSCs after treated with Ms,and increase the endometrium receptivity,and its mechanism may be associated with the activation of autophagy of the hEndoSCs by hUCMSCs-Sup.

Objective To evaluate the relationship between diabetic nephropathy(DN)and diabetic retinopathy(DR)in patients with type 2 diabetes mellitus(T2DM)based on imaging and clinical testing data.Methods Totally 600 T2DM patients who visited the First People's Hospital of Ziyang from March 2021 to December 2022 were included.The fundus photography and fundus fluorescein angiography were performed on all these patients and their age,gender,T2DM duration,cardiovascular diseases,cerebrovascular disease,hypertension,smoking history,drinking history,body mass in-dex,systolic blood pressure,diastolic blood pressure and other clinical data were collected.The levels of fasting blood glu-cose(FPG),triglyceride(TG),total cholesterol(TC),high-density lipoprotein cholesterol(HDL-C),low-density lipo-protein cholesterol(LDL-C),glycosylated hemoglobin(HbA1c),24 h urinary albumin(UAlb),urinary albumin to creati-nine ratio(ACR),serum creatinine(Scr)and blood urea nitrogen(BUN)were measured.Logistic regression was used to analyze the risk factors associated with DR.DR staging was performed according to fundus images,and the convolutional neural network(CNN)algorithm was used as an image analysis method to explore the correlation between DR and DN based on emission computed tomography(ECT)and clinical testing data.Results The average lesion area rates of DR and DN detected by the CNN in the non-DR,mild-non-proliferative DR(NPDR),moderate-NPDR,severe-NPDR and pro-liferative DR(PDR)groups were higher than those obtained by the traditional algorithm(TCM).As DR worsened,the Scr,BUN,24 h UAlb and ACR gradually increased.Besides,the incidence of DN in the non-DR,mild-NPDR,moderate-NPDR,severe-NPDR and PDR groups was 1.67％,8.83％,16.16％,22.16％and 30.83％,respectively.Logistic regression analysis showed that the duration of T2DM,smoking history,HbA1c,TC,TG,HDL-C,LDL-C,24 h UAlb,Scr,BUN,ACR and glomerular filtration rate(GFR)were independent risk factors for DR.Renal dynamic ECT analysis demonstrated that with the aggravation of DR,renal blood flow perfusion gradually decreased,resulting in diminished renal filtration.Conclusion The application of CCN in the early stage DR and DN image analysis of T2DM patients will improve the diag-nosis accuracy of DR and DN lesion area.The DN is worsening as the aggravation of DR.

[摘 要] 目的：设计和构建表达PD-1 shRNA的靶向CD19 CAR-T细胞并验证其体外肿瘤细胞杀伤能力。方法：设计并构建表达PD-1 shRNA的CD19 CAR分子基因，将其包装成逆转录病毒载体，通过qPCR法检测病毒载体拷贝数。将慢病毒转导人原代T细胞，获得三种CAR-T细胞，分别为RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞。采用qPCR法检测三种CAR-T细胞中PD-1 mRNA的表达水平，流式细胞术检测三种CAR-T细胞中PD-1表达水平，萤光素酶报告基因实验、流式细胞术检测在不同效靶比时CAR-T细胞对CD19阳性靶细胞（人淋巴瘤daudi细胞）的杀伤功能。结果：RNAU6-CD19 CAR、PD-1 shRNA1-CD19 CAR、PD-1 shRNA2-CD19 CAR三种CAR分子成功包装成逆转录病毒载体，病毒载体拷贝数均高于1×107拷贝/mL，转导人原代T细胞获得CAR-T细胞，RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞转导效率分别为43.1%、55.1%、41.7%。与RNAU6-CD19 CAR-T细胞相比，PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞中PD-1 mRNA表达水平均显著降低（均P<0.01）、细胞表面PD-1表达水平更低（均P<0.01）、体外对daudi细胞的杀伤率更高（P<0.05或P<0.01）。结论：成功构建表达PD-1 shRNA的靶向CD19 CAR-T细胞，其对CD19阳性靶细胞的杀伤率显著提高，PD-1 mRNA及其翻译产物PD-1的表达减少，CAR-T细胞的耗竭减缓。

Objective:To discuss the effect of Wnt/β-catenin signaling pathway inhibitor methyl 3-｛[(4-methyl-phenyl)sulfonyl]amino ｝ benzoate(MS AB)on the fibrogenic response of the human endometrial stromal cells(HESCs),and to provide the foundation for the application of MSAB in the target therapy of intrauteriue adhesion(JUA).Methods:The normal HESCs were cultured in vitro and divided into two groups:control group and transforming growth factor β1(TGF-β1)group;the HESCs from the adhesion part of the IUA patients were cultured in vitro,regarded as IUA group.Western blotting method was used to detect the expression levels of fibrotic marker protein type Ⅰ collagen α1(COL1A1)in the cells in various groups at different time points(0,12,24,48,and 60 h)after treated with TGF-β1.MTT assay was used to detect the proliferation activities of the cells in various groups.Western blotting method was used to detect the expression levels of the fibrotic marker protein COL1A1,stromal marker proteins such as N-cadherin and α-smooth muscle actin(α-SMA),and Wnt/β-catenin signaling pathway-related protein β-catenin in the cells in control and IUA groups.Based on the MSAB concentrations,the normal HESCs were divided into 0(control),0.25,0.50,0.75,and 1.00 μmol·L-1 MSAB groups,and MTT assay was used to detect the survival rates of the cells in various groups.After treated with MSAB,the normal HESCs were divided into control group(normal HESCs),TGF-β1 group(10 μg·L-1 TGF-β1 induced normal HESCs for 24 h then the drug was withdrawn,replaced with complete culture medium,and the cells continued to be cultured for 24 h),and MSAB group(10 μg·L-1 TGF-β1 induced normal HESCs for 24 h then the drug was withdrawn,replaced with a complete medium containing 0.75 μmol·L-1 MSAB and the cells continued to be cultured for 24 h).Real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of epithelial-mesenchymal transition(EMT)-related transcription factors Snail,Slug,Smuc,ZEB1,and ZEB2,and COL1A1 mRNA in the cells in various groups.Western blotting method was used to detect the expression levels of COL1A1,N-cadherin,α-SMA,β-catenin,and c-myc proteins in the cells in various groups.Results:Compared with control group(after treated with TGF-β1 for 0 h),the expression levels of COL1A1 proteins in the HESCs after treated with TGF-β1 for 12,24,48,and 60 h in TGF-β1 group were increased(P＜0.05 or P＜0.01).Compared with control group,there was no significant difference in the proliferation activity of the HESCs in IUA and TGF-β1 groups(P＞0.05).Compared with control group,the expression levels of COL1A1,β-catenin,N-cadherin,and α-SMA proteins in the cells in IUA group were increased(P＜0.05 or P＜0.01).Compared with control group,the survival rates of the cells in 0.75 and 1.00 μmol·L-1 MSAB groups were decreased(P＜0.05 or P＜0.01).Compared with control group,the expression levels of Snail,Slug,and COL1A1 mRNA in the cells in TGF-β1 group were increased(P＜0.05 or P＜0.01);compared with TGF-β1 group,the expression levels of Snail,Slug,and COL1A1 mRNA in the cells in MSAB group were decreased(P＜0.05 or P＜0.01).Compared with control group,after treated with TGF-β1 for 24 h,the expression levels of COL1A1,N-cadherin,α-SMA,β-catenin,and c-myc proteins in the cells in TGF-β1 group were increased(P＜0.01);compared with TGF-β1 group,the expression levels of COL1A1,N-cadherin,α-SMA,β-catenin,and c-myc proteins in the cells in MSAB group were decreased(P＜0.05 or P＜0.01).Conclusion:MSAB can inhibit the fibrogenic responses of the HESCs in vitro,and the results provide the theoretical basis for the application of MSAB in the target therapy of IUA.

Objective:To investigate the effects of bisphenol A(BPA)on the proliferation activity and stemness characteristics of endometrial mesenchymal stem/stromal cells(eMSCs),and to elucidate the improvement effect of human umbilical cord mesenchymal stem cell-derived supernatant(hUCMSC-Sup)on the cell injury.Methods:The eMSCs were cultured in vitro and treated with different concentrations of BPA(0,200,250,300,350,and 400 μmol·L-1).The eMSCs were divided into control group(only cultured with culture solution),BPA group(cultured with isovolumetric culture solution including 200 μmol·L-1 BPA),BPA+hUCMSC-Sup group(cultured with isovolumetric culture solution including 200 μmol·L-1 BPA and 50%volumetric ratio of hUCMSC-Sup),and BPA+CHIR-99021 group(cultured with isovolumetric culture solution including 200 μmol·L-1 BPA and 10 μmol·L-1 CHIR-99021).The survival rates of eMSCs in various groups were detected by methyl thiazolyl tetrazolium(MTT)assay.The numbers and diameters of the spheroids in various groups were detected by spheroids formation assay,the proliferation activities of the cells in eMSCs stem cell spheroids in various groups were detected by CCK-8 assay;the percentage of CD73+cells in eMSCs in various groups were detected by flow cytometry;the expression levels of sex determining region Y-box 2(Sox2),octamer-binding transcription factor 4(Oct4),and Nanog mRNA in the eMSCs in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)method,the expression levels of β-catenin protein in the eMSCs in various groups were detected by Western blotting method.Results:The MTT results showed that after treated with BPA for 24 and 48 h,compared with 0 μmol·L-1 BPA group,the survival rates of eMSCs in 200,250,300,350,and 400 μmol·L-1 BPA groups were significantly decreased(P<0.01).At 24 and 48 h after treatment,compared with control group,the survival rate of the eMSCs in BPA group was significantly decreased(P<0.01);at 48 h after treatment,compared with BPA group,the survival rate of the eMSCs in BPA+hUCMSC-Sup group was significantly inereased(P<0.05).The spheroids formation assay results showed that compared with culture 3 d group,the numbers and diameters of stem cell spheroids of the eMSCs in culture 4 d group and culture 5 d group were significantly increased(P<0.05 or P<0.01);compared with control group,after 48 h of culture,the number and diameter of the cells in eMSCs stem cell spheroids in BPA group were significantly decreased(P<0.05 or P<0.01).The CCK-8 results showed that after 24 and 48 h of treatment,compared with control group,the proliferation activity of the cells in eMSCs stem cell spheroids in BPA group was significantly decreased(P<0.01);compared with BPA group,the proliferation activity of the cells in eMSCs stem cell spheroids in BPA+hUCMSC-Sup group was significantly increased(P<0.01).The flow cytometry results showed that compared with control group,the percentage of the CD73+cells in the eMSCs in BPA group was significantly decreased(P<0.01);compared with BPA group,the percentage of the CD73+cells in eMSCs in BPA+hUCMSC-Sup group was significantly increased(P<0.01).The RT-qPCR results showed that compared with control group,the expression levels of Sox2,Oct4,and Nanog mRNA in the cells in BPA group were significantly decreased(P<0.01);compared with BPA group,the expression levels of Sox2,Oct4,and Nanog mRNA in the cells in BPA+hUCMSC-Sup group and BPA+CHIR-99021 group were significantly increased(P<0.01).The Western blotting results showed that compared with control group,the expression level of β-catenin protein in the eMSCs in BPA group was significantly decreased(P<0.01);compared with BPA group,the expression levels of β-catenin protein in the eMSCs in BPA+hUCMSC-Sup group and BPA+CHIR-99021 group were signifrcantly inereased(P<0.01).Conclusion:BPA can inhibit the stemness characteristics of the eMSCs,and injury the self-renewal and repair of endometrium;its mechanism may be related to down-regulating the activity of Wnt/β-catenin signal pathway in the cells.hUCMSC-Sup can promote the proliferation of injured eMSCs,and has improvement effect on the stemness injury induced by BPA.

Objective:To analyze the effect of role standardized progressive nursing model on delivery fear and delivery outcome of primiparas.Methods:Using the convenient sampling method, a total of 200 primiparas who were admitted to Ruian Maternal and Child Health Hospital of Zhejiang Province from January to December 2020 were selected as the research objects. According to the random number table method, they were divided into the control group and the observation group, with 100 cases in each group. Patients in the control group were given routine nursing intervention, while patients in the observation group were given role standardization progressive nursing mode on the basis of the control group. Childbirth Attitude Questionnaire (CAQ) and Childbirth Self-Efficacy Inventory-32 (CBSEI-32) were used to investigate, and the outcomes of delivery in the two groups were counted.Results:Before intervention, there was no statistically significant difference in CAQ and CBSEI-32 scores between two groups ( P>0.05) . After intervention, the scores and total scores of all dimensions of CAQ in the observation group were lower than those in the control group, and the outcome expectation, delivery self-efficacy expectation score in CBSEI-32 were higher than those in the control group, and the differences were all statistically significant ( P<0.05) . There was no statistically significant difference in the incidence of postpartum hemorrhage, lateral perineal resection and premature rupture of membranes in the observation group ( P>0.05) . The cesarean section rate in the observation group was lower than that in the control group, and the difference was statistically significant ( P<0.05) . Conclusions:The role standardized progressive nursing model can alleviate the fear of delivery, improve the sense of self-efficacy during delivery and improve the outcome of delivery, which is worthy of clinical application.

Objective:To investigate the influencing factors of hyperuricemia(HUA) and explore early intervention of metabolic diseases.Methods:A total of 70 523 participants were selected from the database of check-ups in 2016. Univariate analysis and logistic regression analysis were used to identify related factors of HUA. Correspondence analysis was performed for the aggregation of different levels of uric acid(UA) and related factors. The mediating effect of mean blood pressure(MBP) between abnormal metabolic indicators and abnormal renal function was tested.Results:The age, sex, occupation, body mass index(BMI), systolic blood pressure, diastolic blood pressure, blood urea nitrogen(BUN), creatinine(Cr), estimated glomerular filtration rate(eGFR), fasting plasma glucose(FPG), total cholesterol(TC), triacylglycerol(TG), high density lipoprotein-cholesterol(HDL-C), low density lipoprotein-cholesterol, plasma viscosity were significantly related to HUA( P<0.001). Logistic regression analysis showed that youth, male, hypertension, TC, TG, and Cr were risk factors for HUA, while HDL-C was a protective factor for HUA( P<0.001). Correspondence analysis showed that during the gradual increase of UA, TC was the first to appear abnormal, followed by hypertension and TG, and the increase of Cr appeared last. Mediating effect showed that in changes of UA, the mediating effects of MBP on TC, TG, and HDL-C were 36.35%, 12.63%, and 9.41%, respectively. In changes of eGFR, the mediating effects of MBP on TC, TG and HDL-C were 30.20%, 27.70%, and 6.13%, respectively. Conclusions:UA is positively correlated with blood pressure, TC, and TG, and inversely with HDL-C. TC and TG have an impact on renal impairment, in which MBP plays a mediating role.