[摘 要] 目的：比较国产与进口CD3/CD28激活磁珠制备的CAR-T细胞的性能差异，为国内CAR-T细胞研发和生产提供备用或替代方案。方法：以进口CD3/CD28激活磁珠︰CD3+ T细胞比例为1∶1的成熟方案作为研究对照，国产磁珠设置1∶2、1∶1和2∶1的比例梯度激活T细胞，T细胞激活72 h后使用CAR慢病毒感染制备CAR-T细胞。分别在慢病毒感染后的第2、4、7天计数，监测CAR-T细胞增殖情况；感染5 d后采用流式细胞术检测CAR-T细胞阳性率；感染8 d后采用流式细胞术检测CAR-T细胞的CD4/CD8分型情况和PD1+ TIM3+耗竭细胞比例。结果：国产CD3/CD28激活磁珠与进口磁珠制备的CAR-T细胞CD4/CD8分型相似，CAR-T细胞阳性率略低于进口磁珠（53.7% vs 57.9%），但CAR-T细胞扩增较进口磁珠增加1倍左右，耗竭水平仅为进口磁珠的一半（4.21% vs 7.91%）。此外，国产磁珠的使用量低于进口磁珠，有利于降低CAR-T细胞研发和生产成本。结论：国产CD3/CD28激活磁珠用于制备CAR-T细胞总体性能不劣于进口磁珠，有潜力作为进口磁珠的备用或替代方案。

Platycodonis Radix is the dry root of Platycodon grandiflorum of Campanulaceae, which has a variety of pharmacological effects and is a commonly used bulk Chinese medicine. In this study, the chloroplast genome sequences of six P. grandiflorum from different producing areas has been sequenced with Illumina HiSeq X Ten platform. The specific DNA barcodes were screened, and the germplasm resources and genetic diversity were analyzed according to the specific barcodes. The total length of the chloroplast genome of 6 P. grandiflorum samples was 172 260-172 275 bp, and all chloroplast genomes showed a typical circular tetrad structure and encoded 141 genes. The comparative genomics analysis and results of amplification efficiency demonstrated that trnG-UCC and ndhG_ndhF were the potential specific DNA barcodes for identification the germplasm resources of P. grandiflorum. A total of 305 P. grandiflorum samples were collected from 15 production areas in 9 provinces, for which the fragments of trnG-UCC and ndhG_ndhF were amplificated and the sequences were analyzed. The results showed that trnG-UCC and ndhG_ndhF have 5 and 11 mutation sites, respectively, and 5 and 7 haplotypes were identified, respectively. The combined analysis of the two sequences formed 13 haplotypes (named Hap1-Hap13), and Hap4 is the main genotype, followed by Hap1. The unique haplotypes possessed by the three producing areas can be used as DNA molecular tags in this area to distinguish from the germplasm resources of P. grandiflorum from other areas. The haplotype diversity, nucleotide diversity and genetic distance were 0.94, 4.79×10^-3 and 0.000 0-0.020 3, respectively, suggesting that the genetic diversity was abundant and intraspecific kinship was relatively close. This study laid a foundation for the identification of P. grandiflorum, the protection and utilization of germplasm resources, and molecular breeding.

A 29-year-old man visited Zhongshan Hospital, Fudan University in December 2021. The patient presented with recurrent coughing, sputum, and wheezing, high level of serum total IgE, positive aspergillus fumigatus-specific IgE and extremely severe mixed ventilatory dysfunction. These features and thoracic CT results scan showed bronchiectasis and allergic bronchopulmonary aspergillosis. In consideration of his clinical characteristics, including low levels of fractional exhaled nitric oxide (FeNO), and nasal nitric oxide (nNO), persistent cough after birth, consanguineous marriage of his parents, etc. we ratiocinated a possibility of hereditary diseases, especially primary ciliary dyskinesia (PCD). From this perspective, whole exome sequencing (WES) was performed and the diagnosis of PCD was ultimately confirmed.

Objective To identify the clinical characteristics and prognostic factors of young patients with sporadic rectal cancer liver metastasis(RCLM).Methods The clinical data of young RCLM patients at 45 years or under(n=40,as younger patient group)in Peking University First Hospital from January 2016 to January 2021 were reviewed,meanwhile,elder RCLM patient group were comprised of 82 patients older than 45-year-old in a 1:2 ratio.Proportions of categorical variables were compared between young patients and old patients.The clinicopathologic parameters were analyzed with univariate and multivariate Cox regression models and Kaplan-Meier method for demonstrating survival differences between the maximum diameter of liver metastasis and local therapy.Results One hundred and twenty-two RCLM patients were identified,the 1-,3-and 5-year survival rates of young patient group were 97.5%,47.5%,15.0%,those of elder patient group were 84.1%,26.8%,9.8%,respectively.The differences in BMI(P=0.008),primary tumor with obstruction and bleeding(P=0.006),synchronous rectal cancer liver metastases(P=0.005),the maximum diameter of liver metastasis>3 cm(P=0.019)were statistically significant between the two groups.And univariate and multivariate analyses showed that age(P=0.003),N stage(P=0.007),local therapy for liver metastases(P=0.047)and the maximum diameter of liver metastasis(P=0.030)were independent risk factors for influencing the prognosis of RCLM patients;curative resection or not of primary tumor(P=0.035)and the maximum diameter of liver metastasis(P=0.041)were independent risk factors for influencing the prognosis of young RCLM patients.Kaplan-Maier curve demonstrated survival differences between the maximum diameter of liver metastasis and local therapy for liver metastasis in RCLM patients(log-rank P=0.000).Conclusions Although with later staging of initial tumor station,young RCLM patients may obtain better survival benefit compared with old patients.Higher degree of lymph node metastasis,local therapy for liver metastases and the maximum diameter of liver metastasis>3 cm indicates poor prognosis in RCLM patients,and without curative resection of primary tumor and maximum diameter of liver metastasis are also considered as the independent poor prognostic factors of young RCLM patients.Local therapy for liver metastases appears to play an important role in the treatment strategy of RCLM patients.

Objective We aim to analyse semaglutide related adverse events in real-world,overall and by gender and age subgroups and compare the differences of different gender and age patients in adverse events,in order to supply references for security usage in the clinic.Methods OpenVigil 2.1 was used to search FDA Adverse Event Reporting System for semaglutide related adverse events from the establishment of the databases to April 2023.According to age and gender,patients were divided into 18-64 years old group and≥65 years old group,male group and female group.We selected ten adverse events which we interested(nausea,diarrhoea,vomiting,pancreatitis,cholecystitis,cholelithiasis,hypoglycaemia,diabetic retinopathy,acute kidney injury and thyroid cancer/medullary thyroid cancer)and analyzed overall and each group of semaglutide adverse events.Results A total of 5 330 cases and 15 558 adverse events were collected.2 935 patients aged 18-64 old group years with 8 553 adverse events;2 395 patients aged≥65 years old group with 7 005 adverse events.2 231 male group patients with 6 195 adverse events;3 059 female group patients with 9 277 adverse events.The sex of 40 patients was unknown.Nausea(1 089 cases/7.00％),vomiting(775 cases/4.98％)and diarrhoea(545 cases/3.50％)remained the most common adverse events.The constituent ratio of pancreatitis was significantly higher in patients aged 18-64 years old group than in patients aged 65 years old group(P＜0.05);the constituent ratio of diarrhoeaand cholelithiasis was significantly lower in patients aged 18-64 years old group than patients aged≥65 years old group(P＜0.05).The constituent ratio of diarrhoea,vomiting,pancreatitis,cholecystitis,cholelithiasis,diabetic retinopathy,and acute kidney injurywas significantly higher in male group patients than in female group(P＜0.05).Conclusion Nausea,diarrhoea and vomiting remined the most common adverse events of semaglutide.Male should be more concerned about gastrointestinal,pancreatitis,gallbladder events,retinopathy and acute kidney injury.Elderly patients should be more alert diarrhoea and cholelithiasis.

Objective To investigate the bioequivalence of gliclazide sustained-release tablets in Chinese healthy subjects.Methods The study was designed using a single-center,open,randomized,single-dose,two-cycle,two-sequence administration method;subjects were orally administered the test/reference preparation 30 mg on an fasting or fed conditions,with self-cross-dosing.The concentration of gliclazide in human plasma was determined by liquid chromatography tandem mass spectrometry(LC-MS/MS)method.The main pharmacokinetic parameters of gliclazide(Cmax,AUC0-t and AUC0-∞)were analyzed by non-atrioventricular model of WinNonlin.Result In the fasting study,24 subjects were recruited and 22 completed the study.The main pharmacokinetic parameters of gliclazide sustained-release tablets test preparation and reference preparation in the fasting group were as follows:Cmax were(862.48±294.48)and(902.96±259.09)ng·mL-1;AUC0-t were(2.60 × 104±8 930.46)and(2.50 ×104±7 573.42)h·ng-1·mL-1;AUC0-∞ were(3.00 × 104±1.43 × 104)and(2.68 × 104±7 085.99)h·ng·mL-1.In the fed study,twenty-four subjects were enrolled and 23 completed the study.The main pharmacokinetic parameters of gliclazide sustained-release tablets test preparation and reference preparation in fed group:Cmax were(1 531.74±273.49)and(1 510.87±241.08)ng·mL-1;AUC0-t were(2.78 ×104±9 565.89)and(2.76 ×104±9 821.43)h·ng·mL-1;AUC0-∞ were(3.02 ×104±1.24 ×104)and(3.02 × 104±1.30 × 104)h·ng·mL-1 h·ng·mL-1.The 90％confidence intervals of the geometric mean ratios of Cmax,AUC0-t,AUC0-∞ for the test preparation and reference preparation gliclazide sustained-release tablets were all between 80％and 125％.Conclusion The test and the reference preparation of gliclazide sustained-release tablets are bioequivalent in Chinese healthy subjects.

Background::Bladder cancer, characterized by a high potential of tumor recurrence, has high lifelong monitoring and treatment costs. To date, tumor cells with intrinsic softness have been identified to function as cancer stem cells in several cancer types. Nonetheless, the existence of soft tumor cells in bladder tumors remains elusive. Thus, our study aimed to develop a microbarrier microfluidic chip to efficiently isolate deformable tumor cells from distinct types of bladder cancer cells.Methods::The stiffness of bladder cancer cells was determined by atomic force microscopy (AFM). The modified microfluidic chip was utilized to separate soft cells, and the 3D Matrigel culture system was to maintain the softness of tumor cells. Expression patterns of integrin β8 (ITGB8), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were determined by Western blotting. Double immunostaining was conducted to examine the interaction between F-actin and tripartite motif containing 59 (TRIM59). The stem-cell-like characteristics of soft cells were explored by colony formation assay and in vivo studies upon xenografted tumor models. Results::Using our newly designed microfluidic approach, we identified a small fraction of soft tumor cells in bladder cancer cells. More importantly, the existence of soft tumor cells was confirmed in clinical human bladder cancer specimens, in which the number of soft tumor cells was associated with tumor relapse. Furthermore, we demonstrated that the biomechanical stimuli arising from 3D Matrigel activated the F-actin/ITGB8/TRIM59/AKT/mTOR/glycolysis pathways to enhance the softness and tumorigenic capacity of tumor cells. Simultaneously, we detected a remarkable up-regulation in ITGB8, TRIM59, and phospho-AKT in clinical bladder recurrent tumors compared with their non-recurrent counterparts.Conclusions::The ITGB8/TRIM59/AKT/mTOR/glycolysis axis plays a crucial role in modulating tumor softness and stemness. Meanwhile, the soft tumor cells become more sensitive to chemotherapy after stiffening, that offers new insights for hampering tumor progression and recurrence.

Objective:To investigate and compare the clinical outcomes of the arterial pre-occlusion technique(APOT) and the traditional technique in the surgery of locally advanced pancreatic cancer with arterial involvement after conversion therapy.Methods:This is a retrospective cohort study. The clinical data of 145 patients with locally advanced pancreatic cancer with arterial involvement admitted to the Department of Hepato-Biliary-Pancreatic Surgery of the First Hospital Affiliated to Naval Medical University,from January 2020 to December 2022 were retrospectively analyzed. All patients completed neoadjuvant therapy for tumors, and the feasibility of radical surgical treatment was determined by a multidisciplinary collaborative team evaluation before surgery. According to whether the intraoperative artery was pre-occluded, 145 patients were divided into two groups, including 28 cases in the APOT group(16 males, 12 females, aged (59.0±9.4) years), and 117 cases in the routine surgery group(76 males, 41 females, aged (55.1±8.2) years). To ensure comparability of baseline data between the APOT group and the routine surgery group, a 1∶2 match was performed using the propensity score matching method, and the caliper value was 0.006 45. The t-test,the Mann-Whitney U test, χ2 test or Fisher′s exact test were used to compare the data between the two groups,respectively. Results:After matching the propensity score,there were 28 cases in the APOT group and 56 cases in the routine surgery group. There were no significant differences in gender,age,preoperative comorbidities,preoperative body mass index,surgical approaches,chemotherapy regimen,stereotactic body radiation therapy ratio,tumor markers,and type of invaded artery between the two groups (all P>0.05).The arterial occlusion time M(IQR) in the APOT group was 7.0(3.8)minutes(range:3 to 15 minutes),and no ischemic manifestations were observed in the distal target organs that blocked blood vessels after surgery. The operation time was (170.3±57.7)minutes in the APOT group and (235.0±80.2)minutes in the routine surgery group,and the difference was statistically significant ( t=-3.800, P<0.01). The APOT group also experienced less intraoperative blood loss(650(588)ml vs. 800(600)ml; U=1 026.500, P=0.021). No significant differences were found between the groups in combined vein resection and reconstruction,celiac trunk resection,early postoperative complications, readmission rates at 30 days,and postoperative length of stay(all P>0.05). Extra-arterial dissection was performed in all patients,with arterial resection and reconstruction in 3 cases: 2 cases in the APOT group(1 case involving the superior mesenteric artery and 1 case involving the common hepatic artery) and 1 case in the routine group(involving the common hepatic artery). Postoperative abdominal bleeding occurred in 4 cases,with 3 cases in the routine group,1 case in the routine group. The R0 resection rate was 85.7%(24/28) in the APOT group and 80.4%(45/56) in the routine group,without significant differences between the groups( P=0.763). The median overall survival time was 27.6 months for the APOT group and 22.5 months for the routine group,while the median disease-free survival was 11.7 months and 16.8 months,respectively,with no significant differences between the two groups( P=0.532, P=0.927). Conclusion:The arterial pre-occlusion technique can be used for extra-arterial dissection in patients with locally advanced pancreatic cancer involving the arteries,reducing surgery time and intraoperative blood loss.

As a potential vectored vaccine,Newcastle disease virus(NDV)has been subject to various studies for vaccine development,while relatively little research has outlined the immunomodulatory effect of the virus in antigen presentation.To elucidate the key inhibitory factor in regulating the interaction of infected dendritic cells(DCs)and T cells,DCs were pretreated with the NDV vaccine strain LaSota as an inhibitor and stimulated with lipopolysaccharide(LPS)for further detection by enzyme-linked immunosorbent assay(ELISA),flow cytometry,immunoblotting,and quantitative real-time polymerase chain reaction(qRT-PCR).The results revealed that NDV infection resulted in the inhibition of interleukin(IL)-12p40 in DCs through a p38 mitogen-activated protein kinase(MAPK)-dependent manner,thus inhibiting the synthesis of IL-12p70,leading to the reduction in T cell proliferation and the secretion of interferon-γ(IFN-γ),tumor necrosis factor-α(TNF-α),and IL-6 induced by DCs.Consequently,downregulated cytokines accelerated the infection and viral transmission from DCs to T cells.Furthermore,several other strains of NDV also exhibited inhibitory activity.The current study reveals that NDV can modulate the intensity of the innate?adaptive immune cell crosstalk critically toward viral invasion improvement,highlighting a novel mechanism of virus-induced immunosuppression and providing new perspectives on the improvement of NDV-vectored vaccine.

Objective To investigate the effects and mechanisms of icariin on changes in fear memory in post-traumatic stress disorder(PTSD)rats.Methods Thirty male SD rats were used to construct a rat model of single prolonged stress(SPS).The model rats were randomly divided into the SPS,icariin,and icariin + K252a(tyrosine kinase receptor B inhibitor)groups(n= 10 each;another 10 normal rats were used as the control group).The icariin and icariin + K252a groups were administered 20 mg/kg icariin by gavage once per day after SPS,while the control and SPS groups were administered the same dose of normal saline.K252a cells were injected into the lateral ventricles.After 2 weeks,anxiety,depression,and fear memory disorder in rats in each group were detected by the mine experiment,ele-vated cross maze experiment,and conditional fear test.The binding activity of icariin to brain-derived neurotrophic factor(BDNF)and the BDNF and TrkB expressions in the rat amygdala were detected by immunohistochemistry.The relative expressions of BDNF and TrkB pro-teins were detected by Western blotting.The expressions of postsynaptic density protein 95(PSD95)and synaptophysin(SYN)in the rat amygdala were detected using immunofluorescence.Results Icariin showed strong binding to BDNF.Compared with the control group,the times of entering the central area and the percentage of movement distance in the central area in the SPS group and the icariin+K252a group were significantly reduced.The open arm entry(OE)and arm opening time(OT)were significantly reduced,the freezing time and defecation times were significantly increased,and the expressions of the BDNF,TrkB,PSD95,and SYN proteins were significantly reduced(P<0.05).Compared with the SPS group,the icariin group rats had significantly increased times of entering the central area and percentages of movement distance in the central area,significantly increased OE and OT,significantly reduced the time of immobilization and defecation,and significantly increased the expressions of BDNF,TrkB,PSD95,and SYN proteins(P<0.05).Conclusion Icariin effectively alleviated the fear memory impairment induced by SPS in rats.This protective effect is related to BDNF/TrkB signaling pathway activation and upregulated PSD95 and SYN expression.