[摘 要] 目的：探讨异位表达血红蛋白亚基（HBA/HBB）对缺氧条件下嵌合抗原受体T细胞（CAR-T细胞）功能障碍的改善作用及其对肿瘤细胞的杀伤效应。方法：全基因合成技术合成靶向HER2的CAR序列，构建共表达HBA或HBB的CAR慢病毒载体，包装慢病毒后感染人原代T淋巴细胞，制备异位表达HBA/HBB的CAR-T细胞，命名为HBA CAR-T和HBB CAR-T。采用缺氧探针检测小鼠实体瘤缺氧状态。通过流式细胞术检测瘤内CAR-T细胞占比、异位表达血红蛋白亚基的CAR-T细胞阳性率及CAR-T细胞的活性氧、凋亡水平。WB法检测HBA CAR-T和HBB CAR-T内相关血红蛋白亚基表达情况，采用细胞计数板计数检测细胞增殖水平，通过萤光素酶报告基因法检测CAR-T细胞对肿瘤细胞的杀伤能力，qPCR检测CAR-T细胞中缺氧诱导因子-1α（HIF-1α）表达水平，利用MitoXpress Intra试剂盒检测CAR-T细胞内氧气含量。结果：不同细胞构建的实体瘤模型均存在明显缺氧情况，且CAR-T细胞浸润水平与缺氧程度呈显著负相关（P < 0.000 1）。HBA CAR-T与HBB CAR-T构建成功（阳性率 > 60%），相应血红蛋白亚基可稳定表达。缺氧环境下HBA CAR-T和HBB CAR-T的ROS水平、凋亡水平显著下降，增殖、对肿瘤细胞的体外杀伤能力显著强于传统CAR-T细胞（均P < 0.05）。HBA CAR-T与HBB CAR-T内HIF-1α表达降低（均P < 0.001），且缺氧程度显著降低（均P < 0.001）。结论：异位表达血红蛋白亚基可改善缺氧条件下CAR-T细胞功能障碍并增强其对肿瘤细胞的体外杀伤作用。

ObjectiveTo investigate how vitamin B₁₂ eye drops regulate MMP-9 and TNF-α expression levels in the corneas of dry eye disease （DED） mouse model induced by a combination of an artificially simulated dry environment （air humidity 30%） and subcutaneous injection of hyoscine hydrobromide. MethodsFifty C57BL/6 female mice were used in the study. 10 mice formed A group （blank control）， while the remaining 40 mice were subjected to dry eye disease induction through subcutaneous injection of scopolamine hydrobromide in a controlled environment with 30% air humidity. All mice underwent schirmer I test （SIT）， tear break-up time （TBUT）， and corneal fluorescence staining score（CFS）evaluations. Mice meeting modeling criteria were randomly assigned to three groups： B group （model group）， C group （PBS control）， and D group （vitamin B₁₂ group）. 0， 7， 14， and 21 days after treatment， each group received TBUT， SIT， CFS， and corneal inflammation assessments. Macroscopic analysis evaluated the efficacy of vitamin B₁₂ eye drops in treating dry eye. After 21 days of treatment， all mice were euthanized via cervical dislocation and their corneas analyzed using HE staining to examine morphological changes. ELISA and immunohistochemical methods were employed to detect MMP-9 and TNF-α expression levels in corneal tissues across groups. ResultsIn dry environments （air humidity 30%）， subcutaneous injection of scopolamine hydrobromide induced changes in dry eye-related indicators in mice： SIT significantly decreased， TBUT significantly shortened， corneal fluorescein staining increased， and CFS all significantly elevated （P 0.05）， indicating successful establishment of the dry eye model. A total of 30 dry eye mice were enrolled. When the dry eye model was successfully established without drug intervention， no statistically significant differences were observed between B group， C group and D group in terms of TBUT， SIT， corneal fluorescein staining， or CFS. Compared with A group， B group， C group and D group showed statistically significant differences in reduced SIT， shortened TBUT， increased corneal fluorescein staining， and elevated CFS （P 0.05）. After drug intervention， D group （vitamin B₁₂ group） exhibited significantly increased SIT， prolonged TBUT， reduced corneal fluorescein staining， clearer corneas， decreased fluorescein staining， and lower CFS compared with B and C groups（P 0.05）. ELISA and immunohistochemical staining results showed that compared with A group， B and C groups exhibited significantly increased expression of MMP-9 and TNF-α in corneal tissues （P 0.05）. D group showed decreased expression of MMP-9 and TNF-α compared with B and C groups but remained higher than A group （P 0.05）. ConclusionThe expression of MMP-9 and TNF-α in the corneal tissues of mice with dry eye is elevated， suggesting that both inflammatory factors， MMP-9 and TNF-α， are involved in the development of dry eye. Vitamin B₁₂ eye drops may improve the symptoms of ocular surface discomfort by promoting the repair of the corneal epithelium. Vitamin B₁₂ eye drops may increase tear secretion， prolong tear film break up time， reduce corneal fluorescein staining， and alleviate the inflammatory reaction of the cornea by decreasing the expression of MMP-9 and TNF-α in the corneal tissues of the mice， thus playing a therapeutic role in the treatment of dry eye disease.

Neuroscience is a significant frontier discipline within the natural sciences and has become an important interdisciplinary frontier scientific field. Brain is one of the most complex organs in the human body, and its structural and functional analysis is considered the “ultimate frontier” of human self-awareness and exploration of nature. Driven by the strategic layout of “China Brain Project”, Chinese scientists have conducted systematic research focusing on “understanding the brain, simulating the brain, and protecting the brain”. They have made breakthrough progress in areas such as the principles of brain cognition, mechanisms and interventions for brain diseases, brain-like computation, and applications of brain-machine intelligence technology, aiming to enhance brain health through biomedical technology and improve the quality of human life. Due to limited understanding and comprehension of neuroscience, there are still many important unresolved issues in the field of neuroscience, resulting in a lack of effective measures to prevent and protect brain health. Therefore, in addition to actively developing new generation drugs, exploring non pharmacological treatment strategies with better health benefits and higher safety is particularly important. Epidemiological data shows that, exercise is not only an indispensable part of daily life but also an important non-pharmacological approach for protecting brain health and preventing neurodegenerative diseases, forming an emerging research field known as motor neuroscience. Basic research in motor neuroscience primarily focuses on analyzing the dynamic coding mechanisms of neural circuits involved in motor control, breakthroughs in motor neuroscience research depend on the construction of dynamic monitoring systems across temporal and spatial scales. Therefore, high spatiotemporal resolution detection of movement processes and movement-induced changes in brain structure and neural activity signals is an important technical foundation for conducting motor neuroscience research and has developed a set of tools based on traditional neuroscience methods combined with novel motor behavior decoding technologies, providing an innovative technical platform for motor neuroscience research. The protective effect of exercise in neurodegenerative diseases provides broad application prospects for its clinical translation. Applied research in motor neuroscience centers on deciphering the regulatory networks of neuroprotective molecules mediated by exercise. From the perspectives of exercise promoting neurogenesis and regeneration, enhancing synaptic plasticity, modulating neuronal functional activity, and remodeling the molecular homeostasis of the neuronal microenvironment, it aims to improve cognitive function and reduce the incidence of Parkinson’s disease and Alzheimer’s disease. This has also advanced research into the molecular regulatory networks mediating exercise-induced neuroprotection and facilitated the clinical application and promotion of exercise rehabilitation strategies. Multidimensional analysis of exercise-regulated neural plasticity is the theoretical basis for elucidating the brain-protective mechanisms mediated by exercise and developing intervention strategies for neurological diseases. Thus,real-time analysis of different neural signals during active exercise is needed to study the health effects of exercise throughout the entire life cycle and enhance lifelong sports awareness. Therefore, this article will systematically summarize the innovative technological developments in motor neuroscience research, review the mechanisms of neural plasticity that exercise utilizes to protect the brain, and explore the role of exercise in the prevention and treatment of major neurodegenerative diseases. This aims to provide new ideas for future theoretical innovations and clinical applications in the field of exercise-induced brain protection.

AIM To study the chemical constituents from the branches and leaves of Michelia yunnanensis Franch.ex Finet & Gagnep.and their anti-inflammatory activities.METHODS The methanol extract was isolated and purified by silica gel,MCI,Sephadex LH-20 and semi-preparative HPLC,then the structures of obtained compounds were identified by physicochemical properties and spectral data.Their anti-inflammatory activities were evaluated by RAW264.7 model.RESULTS Twenty compounds were isolated and identified as dihydrodehydrodiconifenyl alcohol(1),8-hydroxypinoresinol(2),lariciresinol(3),isolariciresinol(4),(7S,8R)-4-hydroxy-3,3',5'-trimethoxy-8',9'-dinor-8,4'-oxyneoligna-7,9-diol-7'-aldehyde(5),thero-2,3-bis-(4-hydroxy-3-methoxypheyl)-3-methoxy-propanol(6),evofolin B(7),(E)-p-coumaryl alcohol γ-O-methyl ether(8),ω-hydroxypropioguaiacone(9),sinapaldehyde(10),isoscopoletin(11),6-hydroxy-5,7-dimethoxycoumarin(12),2α,3α-dihydroxy-2-methylbutyrolactone(13),6-hydroxy-3(1-hydroxy-1-methylethyl)-6-methyl-2-cyclohexen-1-one(14),benzofuran-2-carboxaldehyde(15),3,4-dihydroxy-5-methoxybenzaldehyde(16),3,5-dimethoxy-4-hydroxybenzaldehyde(17),3,4-dihydroxybenzaldehyde(18),3,4-dihydroxybenzoic methyl ester(19),vanillic acid(20).The inhibition rate of compound 1 on NO was 45.39％±0.32％.CONCLUSION Compounds 1-16,18-20 are first isolated from this plant.Compound 1 has anti-inflammatory activity.

OBJECTIVE To observe the impact of Bifidobacterium quadruple viable organisms combined with inter-ferons on serum amyloid A(SAA),transforming growth factor-β(TGF-β),25-hydroxyvitamin D3[25(OH)D3]and intestinal function of the children with rotavirus-induced viral diarrhea.METHODS A total of 88 children with rotavirus-induced viral diarrhea who were treated in Affiliated Hospital of Jiangnan University from Mar.2020 to Dec.2023 were recruited as the research subjects and were randomly divided into the conventional group with 44 cases(conventional symptomatic therapy plus interferons)and the combination group with 44 cases(the conven-tional symptomatic therapy plus Bifidobacterium quadruple viable organisms),and both groups were continuously treated for 5 days.The clinical curative effect and improvement of clinical symptoms were observed and compared between the two groups.The changes of peripheral blood immune function indexes,inflammatory factors,intesti-nal function indexes,SAA,TGF-β and 25(OH)D3 were analyzed.RESULTS There was no significant difference in the effective rate of treatment between the combination group and the conventional group after the treatment for 5 days(Z=1.375,P=0.169).The duration of stool property back to normal,fever abatement,number of defeca-tion times back to normal,cessation of diarrhea,virus turning to be negative and disappearance of abdominal pain was shorter in the combination group than in the conventional group(P＜0.05).The immune function,inflamma-tory factors and intestinal function indexes were improved more remarkably in the combination group than in the conventional group after the treatment for 5 days(P＜0.05).The levels of SAA,TGF-β and 25(OH)D3 of the combination group were respectively(3.84±0.35)pg/ml,(4.87±0.28)pg/ml and(50.63±7.14)pg/ml after the treatment for 5 days,higher than those of the conventional group.CONCLUSION Bifidobacterium quadruple viable organisms combined with interferons can effectively shorten the recovery time of clinical symptoms of the children with rotavirus-induced viral diarrhea,relieve the inflammatory reactions,improve the intestinal function and immune function,and stabilize the levels of serum SAA,TGF-β and 25(OH)D3.

Objective To evaluate the impact of Jixiong Jiedu decoction on the efficacy of diabetic kidney disease in mice and its influence on intestinal flora and trimethylamine oxide(TMAO)levels.Methods Twelve 7-week-old male db/db mice were randomly assigned to the model group or Jixiong Jiedu decoction group(6 mice per group),while 6 male db/m mice were designated as the control group.Following 8 weeks of continuous gavage,we monitored the body weight and blood glucose levels of the mice at weeks 0,4,and 8.Additionally,we assessed urinary microalbumin,kidney injury molecule-1(KIM-1),creatinine(Scr),and urea nitrogen(BUN)levels in urine.Renal pathology was evaluated using HE and PAS staining.Furthermore,fecal samples underwent 16s RNA sequencing,and the serum TMAO levels were determined.Results Compared with the control group,the blood glucose,body weight,8-hour urinary microalbumin,KIM-1 and Scr in the model group were significantly increased,and the renal pathology showed that glomerular segmental mesangial matrix increased,glomerular volume hypertrophy and renal tubular epithelial cell swelling.The abundance of Lactobacillaceae and Lactobacillus in the model group was significantly increased(P<0.01).The abundance of Lachnospiraceae,Helicobacter and Oscillospira decreased significantly(P<0.01),the abundance of each bacterial group changed,and the serum TMAO content increased significantly.Compared with the model group,the 8h urinary microalbumin,KIM-1(P<0.01)and Scr(P<0.05)in the Jixiong Jiedu decoction group were significantly decreased,and there was no significant difference in BUN(P>0.05),and the renal pathological damage was significantly improved.The abundance of Lactobacillaceae and Lactobacillus in intestinal flora decreased significantly(P<0.01),while the abundance of Lachnospiraceae and Oscillospira increased significantly(P<0.01,P<0.05).The structure of gut microbiota,the abundance of dominant and non-dominant bacteria were positively adjusted,and the serum TMAO content was significantly decreased(P<0.01).Conclusion Jixiong Jiedu decoction effectively ameliorates intestinal flora disorders in db/db mice and regulates serum TMAO levels,thereby exerting a nephroprotective effect.

Objective To investigate the distribution and antimicrobial resistance profiles of common pathogens isolated from cerebrospinal fluid(CSF)in CHINET program from 2015 to 2021.Methods The bacterial strains isolated from CSF were identified in accordance with clinical microbiology practice standards.Antimicrobial susceptibility test was conducted using Kirby-Bauer method and automated systems per the unified CHINET protocol.Results A total of 14 014 bacterial strains were isolated from CSF samples from 2015 to 2021,including the strains isolated from inpatients(95.3％)and from outpatient and emergency care patients(4.7％).Overall,19.6％of the isolates were from children and 80.4％were from adults.Gram-positive and Gram-negative bacteria accounted for 68.0％and 32.0％,respectively.Coagulase negative Staphylococcus accounted for 73.0％of the total Gram-positive bacterial isolates.The prevalence of MRSA was 38.2％in children and 45.6％in adults.The prevalence of MRCNS was 67.6％in adults and 69.5％in children.A small number of vancomycin-resistant Enterococcus faecium(2.2％)and linezolid-resistant Enterococcus faecalis(3.1％)were isolated from adult patients.The resistance rates of Escherichia coli and Klebsiella pneumoniae to ceftriaxone were 52.2％and 76.4％in children,70.5％and 63.5％in adults.The prevalence of carbapenem-resistant E.coli and K.pneumoniae(CRKP)was 1.3％and 47.7％in children,6.4％and 47.9％in adults.The prevalence of carbapenem-resistant Acinetobacter baumannii(CRAB)and Pseudomonas aeruginosa(CRPA)was 74.0％and 37.1％in children,81.7％and 39.9％in adults.Conclusions The data derived from antimicrobial resistance surveillance are crucial for clinicians to make evidence-based decisions regarding antibiotic therapy.Attention should be paid to the Gram-negative bacteria,especially CRKP and CRAB in central nervous system(CNS)infections.Ongoing antimicrobial resistance surveillance is helpful for optimizing antibiotic use in CNS infections.

Objective To investigate the antimicrobial resistance of clinical isolates from elderly patients(≥65 years)in major medical institutions across China.Methods Bacterial strains were isolated from elderly patients in 52 hospitals participating in the CHINET Antimicrobial Resistance Surveillance Program during the period from 2015 to 2021.Antimicrobial susceptibility test was carried out by disk diffusion method and automated systems according to the same CHINET protocol.The data were interpreted in accordance with the breakpoints recommended by the Clinical and Laboratory Standards Institute(CLSI)in 2021.Results A total of 514 715 nonduplicate clinical isolates were collected from elderly patients in 52 hospitals from January 1,2015 to December 31,2021.The number of isolates accounted for 34.3％of the total number of clinical isolates from all patients.Overall,21.8％of the 514 715 strains were gram-positive bacteria,and 78.2％were gram-negative bacteria.Majority(90.9％)of the strains were isolated from inpatients.About 42.9％of the strains were isolated from respiratory specimens,and 22.9％were isolated from urine.More than half(60.7％)of the strains were isolated from male patients,and 39.3％isolated from females.About 51.1％of the strains were isolated from patients aged 65-＜75 years.The prevalence of methicillin-resistant strains(MRSA)was 38.8％in 32 190 strains of Staphylococcus aureus.No vancomycin-or linezolid-resistant strains were found.The resistance rate of E.faecalis to most antibiotics was significantly lower than that of Enterococcus faecium,but a few vancomycin-resistant strains(0.2％,1.5％)and linezolid-resistant strains(3.4％,0.3％)were found in E.faecalis and E.faecium.The prevalence of penicillin-susceptible S.pneumoniae(PSSP),penicillin-intermediate S.pneumoniae(PISP),and penicillin-resistant S.pneumoniae(PRSP)was 94.3％,4.0％,and 1.7％in nonmeningitis S.pneumoniae isolates.The resistance rates of Klebsiella spp.(Klebsiella pneumoniae 93.2％)to imipenem and meropenem were 20.9％and 22.3％,respectively.Other Enterobacterales species were highly sensitive to carbapenem antibiotics.Only 1.7％-7.8％of other Enterobacterales strains were resistant to carbapenems.The resistance rates of Acinetobacter spp.(Acinetobacter baumannii 90.6％)to imipenem and meropenem were 68.4％and 70.6％respectively,while 28.5％and 24.3％of P.aeruginosa strains were resistant to imipenem and meropenem,respectively.Conclusions The number of clinical isolates from elderly patients is increasing year by year,especially in the 65-＜75 age group.Respiratory tract isolates were more prevalent in male elderly patients,and urinary tract isolates were more prevalent in female elderly patients.Klebsiella isolates were increasingly resistant to multiple antimicrobial agents,especially carbapenems.Antimicrobial resistance surveillance is helpful for accurate empirical antimicrobial therapy in elderly patients.

OBJECTIVE To enhance the training quality of anticoagulation specialty clinical pharmacists,address the resource limitations of a single training base,and promote homogenization of training quality.METHODS A multi-base joint training system for anticoagulation specialty clinical pharmacists in the Beijing area was established.A mixed research method was employed,collecting data through performance comparisons,questionnaires,and qualitative interviews to compare the differences between the joint training model(experimental group,n=16)and traditional teaching model(the control group,n=17).RESULTS The established joint training system encompassed a unified joint training teaching plan,the formation of a joint training teaching team,the establishment of joint theoretical teaching courses,the implementation of joint case discussions and literature presentations,as well as strengthening the assessment throughout the joint training process.Compared to the control group[theoretical assessment of(76.44±3.66)points,case assessment of(84.31±3.27)points],the experimental group students achieved higher scores in theoretical assessment[(79.85±4.64)points]and case assessment[(88.70±5.51)points](P＜0.05).Through questionnaires and qualitative interviews,the trainees in experimental group were highly satisfied with the joint training model in terms of theoretical learning,communication skills,and teaching interaction.CONCLUSIONS The multi-base collaborative training system for anticoagulation specialty clinical pharmacists can integrate advantageous resources and significantly enhance the training effectiveness of anticoagulation specialty clinical pharmacists,offering value for wider promotion.

Objective:Exploring the role and mechanism of CHMP4C in regulating necroptosis during gastric can-cer development and progression.Method:The expression of CHMP4C in pan-cancer was analyzed by bioinformatics methods,and the expression of CHMP4C was detected in human normal gastric epithelial cells and GC cell lines by RT-qPCR and Western blot.Overexpression or knockdown of CHMP4C was performed in GC cell lines,and the effects of CHMP4C on the growth and proliferation of GC cells were detected using CCK-8 and clone formation assays.The CCK-8 experiment and Hoechst/PI double staining experiment were used to detect the changes in GC cell mortality and PI positive cell ratio after treatment with the necroptsis inducer TSZ or inhibitor necrostatin-1(Nec-1).Western blot assay was used to detect the protein and phosphorylation levels of RIPK1,RIPK3,and MLKL in GC cells.Result:CHMP4C was upregulated in GC tissues and cells.The CCK-8 and clone formation experiments showed that overex-pression of CHMP4C significantly improved the proliferation ability and colony formation efficiency of GC cells,while knockdown of CHMP4C significantly weakened GC cells.Moreover,the results of CCK-8 and Hoechst 33342/PI double staining experiments showed that upregulated CHMP4C could inhibit TSZ induced GC cell death;Nec-1 can reverse the decrease in GC cell viability caused by CHMP4C knockdown.Western blot experiment showed that the levels of p-RIPK1,p-RIPK3,and p-MLKL were significantly decreased in overexpressing cells,while they were increased in knockdown cells.After treatment with Nec-1,the expression levels of these three proteins decreased in knockdown cells.Conclusion:CHMP4C may promote GC progression by negatively regulating necroptosis through inhibiting the phosphorylation of the RIPK1/RIPK3/MLKL signaling pathway,suggesting that it is expected to be a potential target for GC therapy.