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.

Purpose/Significance According to the experience of Sichuan Provincial People's Hospital in implementing the internet outpatient special disease services,the paper puts forward some thoughts and suggestions,so as to provide references for further promoting the application of"internet+"healthcare in outpatient special disease management.Method/Process Based on the operational data of the internet hospital,the paper analyzes the operation status and existing problems of internet outpatient special disease services from the aspects of patient visits,departments,and complaints feedback.Result/Conclusion At present,there are problems such as low utiliza-tion rate of internet outpatient special disease services,poor prescription circulation,and untimely drug delivery.It is necessary to opti-mize online service for key groups,establish the regional electronic prescription sharing platform,improve the drug distribution system,strengthen cooperation between medical institutions at different levels,and build a regional outpatient special disease management system.

Objective To compare the value of interim and end-of-treatment 18F-fluorodeoxyglucose (FDG) PET/CT for the prognosis of patients with diffuse large B-cell lymphoma (DLBCL).Methods A total of 116 DLBCL patients (male ∶female=1 ∶ 1.32,average age:(57.87± 15.89) years) whose initial treatment was rituximab+cyclophosphamide+doxorubicin+vincristine+prednisone(R-CHOP),were enrolled from 2008 to 2016.All patients underwent pre-treatment,interim and end-of-treatment 18F-FDG PET/CT.End points were progression-free survival (PFS) and overall survival (OS).Correlation between metabolic parameters including maximum standardized uptake value (SUVmax),metabolic tumor volume (MTV),total lesion glycolysis (TLG),Deauville score (DS),△SUVmax,△MTV,△TLG in the interim PET/CT and those in the end-of-treatment PET/CT was analyzed with Pearson correlation analysis.The ability of metabolic parameters to assess the prognosis was evaluated by receiver operating characteristic(ROC) curve analysis.The survival was analyzed by Kaplan-Meier method and hazard ratio (HR) was calculated by Cox regression model.Results The r values for △SUVmax,△MTV,△TLG of interim and end-of-treatment PET/CT were 0.630,0.912,0.955,respectively (all P＜0.01).One hundred and four cases (89.7％,104/116)had the same DS in the interim and end-of-treatment PET/CT,and the r value for DS between two PET/CT scans was 0.733 (P＜0.01).The interim metabolic parameters were superior to that at the end of treatment in evaluating the progression.The 5-year PFS (83.1％ vs 35.9％,P＜0.01;HR =5.969,P＜0.01) and OS (95.7％ vs 75.5％,P＜0.01;HR=8.161,P＜0.05) rates were significantly higher in DS 1-3 group than those in DS 4-5 group.Conclusions Interim PET/CT is effective for the prediction of prognosis in DLBCL patients with R-CHOP treatment.Patients with complete remission in the interim can omit the end-of-treatment PET/CT.

Objective·To explore the diagnostic value of 9 mTc-sestamibi (9 mTc-MIBI) single photon emission computed tomography/computed tomography (SPECT/CT) and ultrasonography in hyperparathyroidism. Methods·Fifty patients with hyperparathyroidism were included. 9 mTc-MIBI SPECT/CT was performed before operations in all patients, while ultrasonography was performed in 33 patients. The diagnostic efficiency was calculated for both imaging methods in comparison to pathological data. Results·Serum parathyroid hormone (PTH) levels were 352.0 (141.5-846.0) pg/mL and 1792.0 (1018.5-2358.5) pg/mL, respectively, in primary hyperparathyroidism (PHPT) and secondary hyperparathyroidism (SHPT), while maximum diameters of lesion were 14.5 (9.0-20.9) mm and 10.0 (8.0-12.6) mm, respectively (both P<0.01). The accuracy of SPECT/CT were 97.7% and 62.5%, respectively (P<0.01), in PHPT and SHPT. In 33 patients, the sensitivity and accuracy of SPECT/CT were 66.3% and 74.2%, respectively, whereas the sensitivity and accuracy of ultrasonography were 45.7% and 61.4%, respectively (both P<0.05). Conclusion·Serum PTH levels were higher, while maximum diameters of lesion were longer in PHPT than that in SHPT, and the diagnostic efficiency was also higher in PHPT than that in SHPT. In the other hand, the sensitivity and accuracy of SPECT/CT were higher than that of ultrasonography.

Objective:To investigate the effect of miR-218 on the proliferation and invasion of breast cancer cells.Methods: The expression of miR-218 in breast cancer tissues and breast cancer cell lines was detected by qPCR.The relationship between the expression of miR-218 and the clinicopathological parameters of breast cancer were analyzed.Double luciferase assay was used to detect the relationship between miR-218 and SNX4.MTT assay and invasion assay were used to detect the proliferation and invasion of breast cancer cells after overexpression of miR-218.MTX assay and invasion assay were used to detect the recovery level of SNX4 on the proliferation and invasion of breast cancer cells.The effect of miR-218 on the tumorigenesis of breast cancer cell lines was examined by tumorigenesis in nude mice.Results: The expression level of miR-218 in breast cancer tissue and MCF-7 cell line was higher.The expression of miR-218 was associated with pathological stage of breast cancer and lymph node metastasis.SNX4 may be the target of miR-218.Overexpression of miR-21 could inhibit the proliferation and invasion of breast cancer cells.Overexpression of SNX4 could reverse the inhibitory effect of miR-218 on breast cancer cells.Overexpression of miR-218 could inhibit the breast cancer cell line in nude mice tumorigenic ability.Conclusion: miR-218 can up-regulate the expression of miR-218 in breast cancer,and miR-218 can regulate the expression of SNX4 in breast cancer cell proliferation and invasion.

Objective To investigate the relationship between RAD51 135G ＞ C polymorphism and prognosis in triple-negative breast cancer patients by retrospective analysis.Methods The clinical data of 62 triplenegative breast cancer patients were collected.The 62 cases underwent standard chemotherapy and radiotherapy after tumor resection from Jan.2004 to Dec.2010 in Affiliated Cancer Hospital of Zhengzhou University.RAD51 135G ＞ C polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RELP) technology.The survival curve about progress free and overall survival time were then made.Results The median progress free and overall survival time in triple-negative breast cancer patients with or with-out RAD51 135G ＞ C polymorphism were(77.00 ±5.55)and(89.00 ± 10.40) months vs(99.00 ±4.26)and (103.00 ±4.30) months.The difference had statistical significance(P =0.039 and 0.015 respectively).Conclusion RAD51 135G ＞ C polymorphism is related with prognosis of triple-negative breast cancer patients,which might be a prognostic factor for breast cancer.