Objective:To construct bacterial cytoplasmic membrane nanovesicles(BMV)with overexpressing programmed death 1(PD-1),denoted as BMV-PD-1 and evaluate the targeting efficacy of BMV-PD-1 towards transplanted lung tumor tissues in mice.Methods:The fusion plasmid ClyA-PD-1-EGFP fused by PD-1 and Cytolysin A(ClyA)was transferred into Escherichia coli BL21-Codonplus through plasmid transformation.Laser confocal microscopy,SDS-PAGE,and WB were used to detect the expression of the fusion protein ClyA-PD-1-EGFP.Bacterial membranes were extracted and processed with an extruder to generate BMV-PD-1.TEM and NTA were utilized to assess the morphology,size distribution,and zeta potential of BMV-PD-1,while WB was used to verify the presence of PD-1 protein.Laser confocal imaging was conducted to monitor the uptake of BMV-PD-1 by Lewis lung cancer cells.A C57BL/6J mouse subcutaneous transplant tumor model of LLC lung cancer cells was constructed,and the tumor targeting of BMV-PD-1 was evaluated by small animal imaging system.Results:Laser confocal microscopy images demonstrated that the plasmid ClyA-PD-1-EGFP was transferred into BL21-Codonplus and successfully expressed into protein.SDS-PAGE results suggested that ClyA-PD-1-EGFP was overexpressed in BL21-Codonplus.WB analysis indicated that PD-1 was expressed in bacteria and highly expressed in BMV-PD-1(P<0.001).NTA and TEM analyses revealed that BMV-PD-1 were spherical vesicles with a diameter of(145±14)nm and a negative surface charge.Laser confocal imaging showed that the high expression of PD-1 significantly increased the uptake of BMV-PD-1 by lung cancer cells(P<0.01).In vivo imaging of small animals further confirmed that the high expression of PD-1 can effectively improve cancer targeting of BMV-PD-1(P<0.01).Conclusion:In this study,bacterial plasma membrane nanovesicles BMV-PD-1 with high PD-1 expression are successfully constructed,and it is found that PD-1 overexpression markedly improve the mouse lung cancer xenograft tissue targeting specificity of BMV-PD-1,laying the groundwork for further development of BMV-PD-1 as a carrier for targeted drug delivery systems in tumors.

Objective To explore the anti-depression mechanism of Kai-Xin-San(KXS)via regulation of neurogenesis in hippocampus of depression-like mice.Methods The extracts of KXS were prepared and the anti-depression effects of KXS were evaluated by behavioral tests on chronic unpredictable mild stress(CUMS)induced depression-like mice.Evaluating depression-like behavior in CUMS mice through sucrose preference test,forced swimming test,tail suspension test,and other methods.Neurogenesis in hippocampus were determined by immunofluorescence assay.In addition,effects of KXS on regulating nestin expression and Wnt/b-catenin signaling pathway were explored by western blotting analysis.Amounts of cortisol,corticotropin-releasing factor(CRF),adrenocorticotropic hormone(ACTH),brain-derived neurotrophic factor(BDNF)and nerve growth factor(NGF)were determined by ELISA tests.Mouse primary neural stem cells(NSC)was used to evaluate the effect of KXS on promoting its proliferation by immunofluorescence assay.In addition,effects of KXS on regulating nestin and Wnt/β-catenin signaling pathway were also explored by Western blotting analysis.Results KXS significantly ameliorated the depression-like behaviors in presence of increased sucrose preference rate and decreased immobile time of tail suspension and forced swimming.KXS significantly promoted the neurogenesis in the hippocampus and expressions of nestin,reduced the expressions of cortisol,CRF,ACTH,increased the expressions of BDNF,NGF,and regulated Wnt/β-catenin signaling pathway.KXS also promoted the proliferation of NSCs and expressions of nestin,enhanced the translocation of b-catenin into nucleus,and regulated the expressions of proteins of Wnt/β-catenin signaling pathway.Conclusion KXS promoted neurogenesis in hippocampus and regulated Wnt/β-catenin pathway,which might contribute to its antidepressant effect.

Based on the theory of harmony between man and nature,this article systematically elaborates on the theoretical conno-tation of"adapt to the time"and emphasizes the significant role of daily rhythm,monthly rhythm,annual rhythm and super annual rhythm in female infertility.This paper proposes that female fertility and the implementation of assisted reproductive technology are in-fluenced by time rhythms.It is recognized that irregular menstruation is the key to the onset of female infertility,and the treatment should focus on regulating menstrual cycle.This article further refines the diagnosis and treatment plan of traditional Chinese medicine for female infertility through methods such as hourly,weekly,seasonal and age adjustment,which provides ideas and methods for clini-cal diagnosis and treatment of female infertility and the enlightenment of its medication therapy.