[摘 要] 树突状细胞（DC）作为抗肿瘤免疫应答的核心启动者与调控者，已成为肿瘤免疫治疗的重要靶点。DC疫苗等靶向DC的免疫治疗策略在抗肿瘤治疗中展现出独特优势，但仍存在抗原提呈效率不足、免疫抑制微环境抵抗、功能特异性调控困难等瓶颈问题。肿瘤微环境（TME）中的DC存在高度异质性，不同DC亚群在分化发育、免疫调控及效应转归方面呈现复杂的多样性。解析DC亚群的精确表型与功能机制对于开发新型DC靶向性免疫治疗策略具有重要意义。TME中募集的经典DC、浆细胞样DC、单核细胞来源DC能与肿瘤浸润免疫细胞和微环境中的非免疫细胞（包括肿瘤细胞、成纤维细胞、内皮细胞等）相互作用激活抗肿瘤免疫应答，而TME还会通过转录调控、表观遗传调控、代谢重塑等多种方式抑制DC招募和抗原提呈能力，甚至诱导其向耐受性DC转化。值得注意的是，新近发现的富含免疫调节分子的成熟DC具有双向免疫调控作用，其起源路径和免疫调控网络仍有待深入研究。随着单细胞分析与空间组学分析等技术的发展，有望在单细胞分辨率下系统解析DC亚群的功能多样性及其与TME的时空相互作用网络，为开发下一代精准免疫治疗策略提供新靶点和新思路。

ObjectiveTo analyze the metabolomic characteristics of platelets in fibrinogen（FIB） overexpression rats of coronary heart disease with blood stasis syndrome（CHD-BSS）， explore potential biomarkers， and investigate the mechanism of FIB overexpression on CHD-BSS. MethodsSD rats were randomly divided into BSS group and BSS+FIB overexpression group（BSS+FIB group）， with 10 rats in each group. Both the BSS+FIB group and the BSS group were fed a high-fat diet combined with oral administration of vitamin D₃ and subcutaneous injection of isoproterenol， but rats in the BSS+FIB group were overexpressed with FIB during the initial modeling stage by transfection with adeno-associated virus（AAV）. The overexpression level of FIB in rat liver and plasma samples was detected by enzyme-linked immunosorbent assay（ELISA） and real-time fluorescence quantitative polymerase chain reaction（Real time PCR）， as well as the expression level of liver FIB A（FGA） mRNA. The characteristics of metabolites in rat platelet samples were analyzed by ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap-MS）， and the differential metabolites between groups were screened by principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA）， and the enriched pathways were analyzed. The accuracy of potential biomarkers in the diagnosis of CHD-BSS was evaluated by receiver operating characteristic（ROC） curve. The expression of autophagy related proteins phosphorylated adenosine monophosphate（AMP） activated protein kinase（p-AMPK）/AMPK， phosphorylated mammalian target of rapamycin（p-mTOR）/mTOR， microtubule-associated protein 1 light chain 3（LC3） Ⅱ/Ⅰ and p62 in platelets were detected by Western blot. ResultsCompared with the BSS group， the expression levels of FIB in liver and plasma samples of the BSS+FIB group were significantly increased（P<0.05， P<0.01）， and the expression level of FIB mRNA in the liver was remarkably increased（P<0.01）， indicating successful overexpression of FIB. Platelet metabolomics results showed significant differences in metabolic profiles between the BSS+FIB group and the BSS group， and a total of 25 significantly enriched metabolic pathways and 8 metabolites involved in these metabolic pathways， among which uric acid， guanosine and ribose 1-phosphate levels were up-regulated， while adenosine diphosphate（ADP）， AMP， guanosine diphosphate（GDP）， adenylosuccinate and norepinephrine levels were down-regulated. The diagnostic ability analysis of differential metabolites showed that all 8 differential metabolites had good diagnostic ability， with an area under the curve（AUC）>0.85. Western blot results showed that compared with the BSS group， the expression levels of p-mTOR/mTOR and p62 proteins in platelets of the BSS+FIB group was significantly reduced（P<0.01）， while the expression levels of p-AMPK/AMPK and LC3Ⅱ/Ⅰ proteins were increased， but the difference was not statistically significant. ConclusionOverexpression of FIB can change the metabolic characteristics of CHD-BSS rat model， involving multiple aspects such as vascular endothelial injury， platelet activation and myocardial function damage. Among them， overexpression of FIB may enhance the occurrence of platelet autophagy， thereby inducing platelet activation and promoting thrombus formation.

Repeated transcranial magnetic stimulation (rTMS) is one of the commonly used brain stimulation techniques. In order to investigate the effects of rTMS on the excitability of different types of neurons, this study is conducted to investigate the effects of rTMS on the cognitive function of mice and the excitability of hippocampal glutaminergic neurons and gamma-aminobutyric neurons from the perspective of electrophysiology. In this study, mice were randomly divided into glutaminergic control group, glutaminergic magnetic stimulation group, gamma-aminobutyric acid energy control group, and gamma-aminobutyric acid magnetic stimulation group. The four groups of mice were injected with adeno-associated virus to label two types of neurons and were implanted optical fiber. The stimulation groups received 14 days of stimulation and the control groups received 14 days of pseudo-stimulation. The fluorescence intensity of calcium ions in mice was recorded by optical fiber system. Behavioral experiments were conducted to explore the changes of cognitive function in mice. The patch-clamp system was used to detect the changes of neuronal action potential characteristics. The results showed that rTMS significantly improved the cognitive function of mice, increased the amplitude of calcium fluorescence of glutamergic neurons and gamma-aminobutyric neurons in the hippocampus, and enhanced the action potential related indexes of glutamergic neurons and gamma-aminobutyric neurons. The results suggest that rTMS can improve the cognitive ability of mice by enhancing the excitability of hippocampal glutaminergic neurons and gamma-aminobutyric neurons.
Animals ; Mice ; Hippocampus/cytology* ; Transcranial Magnetic Stimulation ; Neurons/physiology* ; Male ; Cognition/physiology* ; gamma-Aminobutyric Acid/metabolism* ; Action Potentials/physiology*

Objective To explore the effect and preliminary mechanism of the plant-derived immunostimulatory molecule,ophiopogonin,on enhancing the immune response of a subunit vaccine with the receptor-binding domain(RBD)of coronavirus spike protein as the antigen.Methods CCK-8 assay was used to determine the cytotoxicity of ophiopogonin D'(OPD')on bone marrow-derived dendritic cells(BMDCs).Female Balb/c mice were randomly divided into RBD,RBD/OPD',RBD/Alum,and control groups.The immunization dose was 5 μg of antigen per mouse and 100 μg of adjuvant per mouse,and immunization was carried out according to the intramuscular injection immunization procedure on days 0,21,and 42.The titers of specific IgG and its subtype antibodies were detected by ELISA.The cytokine levels in the supernatant of splenocytes were detected using ELISA.The number of splenocytes secreting IFN-γ was detected by ELISpot.Laser confocal microscopy was employed to observe the uptake of antigen by BMDCs.The phagocytic ability of BMDCs for antigen was quantitatively analyzed by flow cytometry.The mechanism of its enhanced immune effect was preliminarily explored using transcriptomics technology combined with bioinformatics research.Results When the concentration of OPD'was less than 5 μg/mL,the survival rate of BMDCs was 100%.After a single intramuscular injection in mice,except for a slight decrease in body weight,the other biochemical indicators were within corresponding normal ranges.After intramuscular injection immunization of the vaccine,the titers of serum-specific IgG,IgG1,and IgG2a in the RBD/OPD'group were significantly higher than those in the RBD group(P<0.05).Compared with the RBD group,the RBD/OPD'group induced a high-level Th1 cell immune response of IL-1β,TNF-α,and IFN-γ(P<0.01)and had more lymphocytes secreting IFN-γ(P<0.001).Laser confocal microscopy displayed that BMDCs took up more antigens after OPD'treatment,which was further confirmed with flow cytometry in quantitative analysis on antigen uptake rate(P<0.01).Transcriptomics results indicated that there was more significant enrichment of the PPAR signaling pathway in the RBD/OPD'group than the RBD group,suggesting that OPD'may activate the PPAR signaling pathway to exert its adjuvant effect.Conclusion OPD'effectively enhances the immune response of the RBD subunit vaccine,and its action mechanism may be related to the activation of the PPAR signaling pathway.

Objective To establish a mouse model of infection with the minimum lethal dose of Vibrio vulnificus(V.vulnificus)and to evaluate the protective efficacy of inactivated whole-cell(IWC)vaccine using this model.Methods A mouse model of lethal-dose infection was established by intraperitoneal injection of different doses of V.vulnificus.Bacterial colonization in the organs was detected with tissue homogenate plating,and pathological changes in the organs were observed after tissue section staining.Flow cytometry was used to detect immune cell responses after liver tissues were digested into single-cell suspension.IWC vaccine of V.vulnificus was prepared,and the mice were immunized through different routes to observe the protective efficacy of the vaccine.Results A mouse model of infection with the minimum lethal dose at 1×106 CFU of V.vulnificus was successfully established.After infection,the bacteria were mainly colonized in the liver of mice and caused severe pathological damages.Compared with the uninfected mice,the proportion of neutrophils in the liver was significantly increased in the infected mice,whereas the proportions of B cells and T cells were correspondingly decreased(P<0.05).A single intramuscular or intraperitoneal injection of the IWC vaccine could protect the mice effectively against lethal infection of V.vulnificus in 7 d later(P<0.01),although the level of serum IgG having no significant increase.Conclusion A mouse model of lethal-dose infection with V.vulnificus is successfully established,with histopathological characteristics.The IWC vaccine of V.vulnificus rapidly mediates immune protection in this model probably independent of IgG.

Objective To explore the mechanism underlying the protective effect of α2-macroglobulin (A2M) against glucocorticoid-induced femoral head necrosis. Methods In a human umbilical vein endothelial cell (HUVEC) model with injuries induced by gradient concentrations of dexamethasone (DEX;10-8-10-5 mol/L), the protective effects of A2M at 0.05 and 0.1 mg/mL were assessed by examining the changes in cell viability, migration, and capacity of angiogenesis using CCK-8 assay, Transwell and scratch healing assays and angiogenesis assay. The expressions of CD31 and VEGF-A proteins in the treated cells were detected using Western blotting. In BALB/c mouse models of avascular necrosis of the femoral head induced by intramuscular injections of methylprednisolone, the effects of intervention with A2M on femoral trabecular structure, histopathological characteristics, and CD31 expression were examined with Micro-CT, HE staining and immunohistochemical staining. Results In cultured HUVECs, DEX treatment significantly reduced cell viability, migration and angiogenic ability in a concentration- and time-dependent manner (P<0.05), and these changes were obviously reversed by treatment with A2M in positive correlation with A2M concentration (P<0.05). DEX significantly reduced the expression of CD31 and VEGF-A proteins in HUVECs, while treatment with A2M restored CD31 and VEGF-A expressions in the cells (P<0.05). The mouse models of femoral head necrosis showed obvious trabecular damages in the femoral head, where a large number of empty lacunae and hypertrophic fat cells could be seen and CD31 expression was significantly decreased (P<0.05). A2M treatment of the mouse models significantly improved trabecular damages, maintained normal bone tissue structures, and increased CD31 expression in the femoral head (P<0.05). Conclusion A2M promotes proliferation, migration, and angiogenesis of DEX-treated HUVECs and alleviates methylprednisolone-induced femoral head necrosis by improving microcirculation damages and maintaining microcirculation stability in the femoral head.

Objective To explore the mechanism underlying the protective effect of α2-macroglobulin (A2M) against glucocorticoid-induced femoral head necrosis. Methods In a human umbilical vein endothelial cell (HUVEC) model with injuries induced by gradient concentrations of dexamethasone (DEX;10-8-10-5 mol/L), the protective effects of A2M at 0.05 and 0.1 mg/mL were assessed by examining the changes in cell viability, migration, and capacity of angiogenesis using CCK-8 assay, Transwell and scratch healing assays and angiogenesis assay. The expressions of CD31 and VEGF-A proteins in the treated cells were detected using Western blotting. In BALB/c mouse models of avascular necrosis of the femoral head induced by intramuscular injections of methylprednisolone, the effects of intervention with A2M on femoral trabecular structure, histopathological characteristics, and CD31 expression were examined with Micro-CT, HE staining and immunohistochemical staining. Results In cultured HUVECs, DEX treatment significantly reduced cell viability, migration and angiogenic ability in a concentration- and time-dependent manner (P<0.05), and these changes were obviously reversed by treatment with A2M in positive correlation with A2M concentration (P<0.05). DEX significantly reduced the expression of CD31 and VEGF-A proteins in HUVECs, while treatment with A2M restored CD31 and VEGF-A expressions in the cells (P<0.05). The mouse models of femoral head necrosis showed obvious trabecular damages in the femoral head, where a large number of empty lacunae and hypertrophic fat cells could be seen and CD31 expression was significantly decreased (P<0.05). A2M treatment of the mouse models significantly improved trabecular damages, maintained normal bone tissue structures, and increased CD31 expression in the femoral head (P<0.05). Conclusion A2M promotes proliferation, migration, and angiogenesis of DEX-treated HUVECs and alleviates methylprednisolone-induced femoral head necrosis by improving microcirculation damages and maintaining microcirculation stability in the femoral head.

In recent years, studies have shown that transplanted neural stem cells (NSCs) help neural tissues regenerate and return to normal through paracrine action rather than just replacing cells. Exosomes are essential paracrine mediators that can participate in cell communication through substance transmission. This review focuses on NSCs regulated by exosomes and their application in treatment of nervous system diseases, in order to provide important references for further research and clinical application of NSCs exosomes..

Objective:To understand the current situation of postpartum maternal perceived spouse support for breastfeeding, and explore its mediating role in the spouse support for breastfeeding and maternal breastfeeding self-efficacy.Methods:From October 2021 to October 2022, convenience sampling was used to select 602 postpartum women (42 days postpartum) and their spouses who underwent follow-up visits at Obstetric Clinics in four ClassⅢ hospitals in Shanghai as the research subject. A questionnaire survey was conducted using the General Information Questionnaire, Partner Breastfeeding Influence Scale (PBIS) , and Breastfeeding Self-efficacy Scale Short Form (BSES-SF) .Results:The PBIS score of 602 postpartum women was (121.07±25.41) , and the PBIS score of 602 maternal spouses was (134.37±18.94) . Pearson correlation analysis results showed that maternal perceived spouse support has a positive correlation with spouse support ( P<0.01) , and a positive correlation with maternal self-efficacy in breastfeeding ( P<0.01) ; spouse support was positively correlated with maternal self-efficacy in breastfeeding ( P<0.01) . The mediating effect analysis that maternal perceived spouse support played a complete mediating role between spouse support and maternal self-efficacy in breastfeeding. Conclusions:There are both connections and differences between maternal perceived spouse support and spouse support, and the positive promoting effect of spouse support on maternal self-efficacy in breastfeeding can be completely mediated by maternal perception. Medical and nursing staff should pay attention to the assessment of maternal perceived spouse support and guide spouse breastfeeding support behavior based on maternal needs and experiences of spouse support, so as to improve breastfeeding outcomes.

Although multifarious tumor-targeting modifications of nanoparticulate systems have been attempted in joint efforts by our predecessors, it remains challenging for nanomedicine to traverse physiological barriers involving blood vessels, tissues, and cell barriers to thereafter demonstrate excellent antitumor effects. To further overcome these inherent obstacles, we designed and prepared mycoplasma membrane (MM)-fused liposomes (LPs) with the goal of employing circulating neutrophils with the advantage of inflammatory cytokine-guided autonomous tumor localization to transport nanoparticles. We also utilized in vivo neutrophil activation induced by the liposomal form of the immune activator resiquimod (LPs-R848). Fused LPs preparations retained mycoplasma pathogen characteristics and achieved rapid recognition and endocytosis by activated neutrophils stimulated by LPs-R848. The enhanced neutrophil infiltration in homing of the inflammatory tumor microenvironment allowed more nanoparticles to be delivered into solid tumors. Facilitated by the formation of neutrophil extracellular traps (NETs), podophyllotoxin (POD)-loaded MM-fused LPs (MM-LPs-POD) were concomitantly released from neutrophils and subsequently engulfed by tumor cells during inflammation. MM-LPs-POD displayed superior suppression efficacy of tumor growth and lung metastasis in a 4T1 breast tumor model. Overall, such a strategy of pathogen-mimicking nanoparticles hijacking neutrophils in situ combined with enhanced neutrophil infiltration indeed elevates the potential of chemotherapeutics for tumor targeting therapy.