Objective:To prepare the bacterial outer membrane vesicles(OMV)that can express programmed death ligand 1(PD-L1)nanobody on surface,and to discuss its structural characteristics,cell compatibility,intracellular distribution,and its blocking effect on the programmed cell death protein-1(PD-1)/PD-L1 signaling axis.Methods:The pET28a-ClyA-PD-L1nb prokaryotic expression vector was constructed and transformed into Escherichia coli BL21(DE3)competent cells;the OMV was isolated from the BL21(DE3)monoclonal colonies transformed with the PD-L1nb expression vector by ultracentrifugation;the protein purification was performed using the histidine(His)tag;transmission electron microscope and nanoparticle size analyzer were used to analyze and identify the OMV;the OMV isolated from the BL21(DE3)monoclonal colonies transformed with the PD-L1nb expression vector was used as experimental group;the OMV isolated from the untransformed BL21(DE3)monoclonal colonies was used as control group;Western blotting method was used to detect the expression levels of ClyA-PD-L1nb fusion protein in the OMV in two groups;cell counting kit-8(CCK-8)assay was used to detect the activities of mouse macrophage RAW 264.7 cells,mouse triple-negative breast cancer 4T1 cells,and human embryonic kidney HEK293T cells after treated with OMV;fluorescence imaging technology was used to observe the tumor cell endocytosis of OMV;flow cytometry was used to detect the binding effect of OMV to the PD-L1 on surface of the tumor cells in PBS group,OMV-PD-L1nb group,and aPD-L1+OMV-PD-L1nb group.Results:The sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)results showed that after induction of Escherichia coli,significantly thickened protein bands appeared near the predicted relative molecular mass(about 49 000),and after purification,no obvious impurity proteins existed in the lanes;the OMV-PD-L1nb with a size of about 120 nm was isolated by ultracentrifugation,and it presented a uniform spherical structure under transmission electron microscope;the Western blotting results showed that the specific band of ClyA-PD-L1nb was detected in the OMV in experimental group;the CCK-8 assay results showed that after treated with different concentrations of OMV,the viabilities of the RAW 264.7 cells,4T1 cells,and HEK293T cells were all close to 100％;the fluorescence imaging results showed that OMV-PD-L1nb was endocytosed by 4T1 cells and dispersed in the cytoplasm;compared with OMV-PD-L1nb group,the average fluorescence intensity in the cells in aPD-L1+OMV-PD-L1nb group was significantly decreased(P＜0.001).Conclusion:The OMV surface-displaying PD-L1nb,OMV-PD-L1nb,is successfully prepared and isolated;OMV-PD-L1nb shows good compatibility on mouse macrophage cells,tumor cells,and human embryonic kidney cells,can be endocytosed by tumor cells,and successfully blocks the PD-1/PD-L1 signaling pathway.

Objective:To investigate the effect of HSM on the inflammation and pulmonary fibrosis in mice with cecal ligation and puncture ( CLP) . Methods: Both 5 day and 10 day timepoint pulmonary fibrosis were induced by CLP;mice were randomly divided into three groups,including sham group (sham,n=5),model group (CLP,n=11),therapy group (HSM,n=11). HSM extract (200 mg/kg,less than human dose of 0. 27 g/kg) was orally administered to HSM group 2 hour before surgery and repeated everyday, while sham group and model group were given the same dose of normal saline. We used Q-PCR assay to analyze the expression of in-flammatory molecules ( IL-1β and TNF-α) and fibrogenic cytokines ( TGF-β, MMP9 and TIMP1 ) from lung tissues , we used FACS assay to analyze the amount of Th1 cells in PBMC and spleen sections,as well as Treg in spleen sections. Besides,lung sections were subjected to HE stain and immunohistochemical staining withα-SMA and fibronectin. Results:The amount of model group's Th1 cells in PBMC and spleen sections,as well as Treg in spleen sections were significantly lower than sham group's,whereas HSM succeeded in raising them. By day 5, the expression of inflammatory molecules IL-1β and fibrogenic cytokines (TGF-β,MMP9 and TIMP1) from CLP group's lung sections were remarkably improved,by day 10,the expression had decreased but was higher than sham group's;HSM inhibited these cytokines' expression. The expression of TNF-α of CLP group and HSM group had no significant changes. The CLP group's HE results showed obvious pulmonary inflammatory damage,by day 10,the situation had improved,whereas HSM reduced the histopathologic alterations;contrast to sham group,model group's expression of α-SMA and fibronectin was remarkably improved,while HSM group showed lower expression. Conclusion: HSM extract can suppress inflammation, balances immune system and relieves pulmonary fibrosis.

Objective:To investigate the effect of HSM on the immunoreaction and renal fibrosis in mice with cecal ligation and puncture (CLP).Methods: Renal fibrosis was induced by CLP;mice were divided into three groups,including sham group (sham,n =5),model group (CLP,n =11),therapy group (HSM,n =11).HSM extract [200 mg/(kg?d)] was orally administered to HSM group2 hour before surgery and repeated everyday throughout 10 days,while sham group and model group were given the same dose of normalsaline.FACS assay was used to analyze the amount of macrophages ,neutrophils and Treg in PBMC,as well as macrophages in peritonealfluid;we used Q-PCR assay to analyze the expression of inflammatory molecules (IL-1βand TNF-α) and fibrogenic cytokines (TGF-β,MMP9 and TIMP1) from renal sections.Besides,renal sections were subjected to HE stain and immunohistochemical staining with α-SMA and fibronectin.Results: The amount of model group′s macrophages and neutrophils in PBMC,as well as macrophages in theperitoneal fluid were significantly higher than sham group ′s,whereas HSM succeeded in lowering them;contrast to sham group,Tregs′amount of CLP group and HSM group in PBMC had no significant changes .The expression of inflammatory molecules (IL-1βand TNF-α) and fibrogenic cytokines (TGF-β,MMP9 and TIMP1) from CLP group′s renal sections were remarkably improved ,whereas HSM inhibitedthat.The CLP group′s HE results showed obvious renal inflammatory damage ,whereas HSM reduced the histopathologicalterations;contrast to sham group,model group′s expression of α-SMA and fibronectin was remarkably improved,while HSM groupshowed lower expression.Conclusion: HSM extract could regulate immunity response and had effect in improving renal fibrosis .

Objective To investigate the clinical significance of beta-receptor blocker intervention on ECG changes in climac-teric women.Methods Sixty-two subjects of climacteric women without organic diseases but with ECG changes were chosen and ran-domly divided into 2 groups:the control group(n=32)and the treatment group(n=30).The subjects of the control group were not giv-en any treatment, while the subjects of the treatment group were given treatment of beta-receptor blocker intervention for 3 weeks.ECG changes ( heart rate, rhythm and ST-T changes) were detected both before and after treatment, and then, comparisons were made be-tween the groups and within the groups.Results ( 1 ) After 3 weeks of beta-receptor blocker intervention, significant ECG changes could be noted in heart rate, rhythm and ST-T changes ( except ST cable) in the subjects of the treatment group, with statistical signifi-cance, when comparisons were made between the 2 groups (P<0.05).(2)In treatment group, though changes were seen in the paired atrial premature beat ( APB) , ST cable and T wave inversion, there no statistic significance, when comparisons were made between pre and post treatment (P>0.05).There were significant changes in the remaining parameters, with statistical significance (P<0.05). Conclusion Beta-receptor blocker had its clinical value, when it was used in climacteric women with ECG changes.

As the specific endangered medicinal plant in Xinjiang, resources and distribution of Ferula sinkiangen-sis are important for biodiversity conservation and sustainable development of Chinese medicine resources. The spa-tial distribution and resources of F. sinkiangensis were researched based on low altitude remote sensing and sample investigation. The results showed that the optimum working time for F. sinkiangensis monitoring by low altitude remote sensing was the nearby 5 hills, which covered about 0.88 km2. It was suggested that the fence area should be expanded for protection. According to the results of low altitude remote sensing, the amount of F. sinkiangensis in yellow (diameter exceeding 0.3 m) was about 3 191. However, the sample investigation on amount of F. sinkiangensis in yellow (diameter exceeding 0.3 m) was about 2 752. The error between them was 14%. The monitoring time and range for F. sinkiangensis by low altitude remote sensing were also ensured. It was concluded that low altitude re-mote sensing had the advantage of quickly receiving distribution situation of F. sinkiangensis, which can effectively evaluate dynamic changes of F. sinkiangensis in Xinjiang.

Objective To establish the HPLC fingerprints for the guality control of ascidian Styela plicata.Methods The HPLC fingerprints of ten batches of samples were obtained using ZORBAX Bonus-RP(4.6?250mm,5?m) column with a mix of acetonitrile and water with 0.01%TFA as mobile phase in a gradient mode,the detection wavelength was 254nm and the temperature was 30℃.Results The experiment and analysis were carried out on ten samples,the standard HPLC fingerprint pattern method and 13 characteristic diffraction peaks of Styela plicata were obtained.The proposed method was precise,reproducible and steady.Conclusion This reliable and convenient method can be used for the identification and quality control of Styela plicata.