Objective:To prepare the nanodrug paclitaxel dimer(PTX2)-loaded nanoparticles(NPs)using the block copolymer 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol 2000,(DSPE-PEG2000),and to explore the killing effect of PTX2 NPs on the human lung cancer A549 cells and its influence on apop to tis.Methods:The PTX2 NPs were prepared using nanoprecipitation method.Dynamic light scattering(DLS)was employed to determine the particle size distribution,and transmission electron microscope(TEM)was used to observe the ultrastructure of the nanoparticles.After treatment of 0 and 10 mmol·L-1 dithiothreitol(DTT),dialysis method was used to evaluate the in vitro drug release profile of PTX2 NPs.The cell counting kit-8(CCK-8)method was used to assess the survival rates of the A549 cells after treated with PTX2 and PTX2 NPs with different concentrations(0.000 1,0.001 0,0.010 0,0.100 0,and 1.000 0 μmol·L-1).The A549 cells were divided into control group,PTX2 group,and PTX2 NPs group.Live/dead staining method was used to detect the survival of the A549 cells in various groups,and flow cytometry was used to detect the apoptotic rates of the A549 cells in various groups.Results:The mean hydrodynamic diameter of PTX2 NPs was determined to be 144.7nmbyDLS.TheTEM imaging confirmed uniform spherical morphology of PTX2 NPs.In a reductive environment,the PTX2 NPs exhibited continuous drug release with total paclitaxel(PTX)release of 84％within 72 h.The results of CCK-8 method showed that both PTX2 and PTX2 NPs inhibited the proliferation of A549 cells in a dose-dependent manner.When the concentrations of PTX＜0.01 μmol·L-1,compared with PTX2 group,the survival rates of A549 cells in PTX2 NPs group were significantly decreased(P＜0.01 or P＜0.001).The live/dead staining results showed that compared with PTX2 group,the number of red fluorescence-labeled dead cells in PTX2 NPs group was increased.The flow cytometry results demonstrated that compared with control group and PTX2 group,the apoptotic rates of the A549 cells in PTX2 NPs group were significantly increased(P＜0.05 orP＜0.01).Conclusion:The PTX2-loaded nanoparticles PTX2 NPs are successfully prepared which exhibits responsive drug release and demonstrates a more significant killing effect on the human lung cancer A549 cells compared to PTX2.

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 long-term survival and safety in patients with muscle-invasive bladder cancer (MIBC) who experienced a noninvasive down-staging (≤pT 1)after transurethral resection of bladder tumor (TURBT) plus systemic chemotherapy and received bladder-sparing treatment. Methods:The records of patients with MIBC who underwent maximal TURBT plus systemic chemotherapy-guided bladder-sparing treatment were reviewed retrospectively from Dec 2013 to Dec 2020. Eventually, 22 patients who achieved noninvasive down-staging underwent conservative management. The total patient cohort contained 10 males and 12 females. A majority of patients had single lesion and stage T2 disease. The median age of the patients was 66 years and the median tumor size was 3.0 cm. All patients underwent maximal TURBT to resect all visible diseases and followed by 3-4 cycles platinum-based systemic chemotherapy. After achieving noninvasive down-staging, 14 patients received concurrent chemoradiotherapy, and the other 8 patients underwent surveillance. Overactive bladder symptom score (OABSS) was used to assess the bladder function after treatment.Results:Twelve patients achieved pT 0 and 10 patients were down-staged to cT a-T 1. At a median follow-up of 36.7 months, 90.9%(20/22) patients retained their bladder function successfully. Among the 14 patients who received concurrent chemoradiotherapy, 4 had grade 3 or 4 adverse events. Among the 8 patients who underwent surveillance, 3 had grade 3 or 4 adverse events after systemic chemotherapy.Nine patients experienced tumor recurrence in the bladder, and 2 patients died of bladder cancer. Seven (31.8%) patients experienced Ⅲ/Ⅳ grade complications. The 5-year recurrence-free survival (RFS) and overall survival (OS) in patients achieved pT0 were 66.7% and 100.0%, respectively. The 5-year RFS and OS in patients achieved cTa-T1 were 40% and 72%, respectively. The OABSS score of 20 patients who retained their bladder successfully was (1.00±1.03). Conclusions:MIBC patients who achieved noninvasive down-staging might be candidates for the bladder-sparing treatment with maximum TURBT followed by systemic chemotherapy.The patients who achieved pT 0 might have better prognosis with functional bladder.

Objective:To investigate the relationship between antibacterial treatment scheme and prognosis, and to analyze the mortality risk factors of bloodstream infection with carbapenem-resistant Klebsiella pneumoniae(CRKP).Methods:A retrospective case-control study was conducted. The CRKP isolated from clinical venous blood samples in the First Medical Center, Chinese PLA General Hospital between January 1, 2013 and December 31, 2018(not included from January 1, 2016 to December 31, 2017) was collected. According to relevant standards, a total of 50 patients with bloodstream infection with CRKP were included. The patients were divided into death (19 cases) or survival (31 cases) group according to their hospitalization outcomes, and clinical data and antibacterial treatment scheme after infection were collected. The clinical features of the two groups and the correlation between different antibacterial treatment regimens and prognosis were compared. Logistics regression model was used to analyze the risk factors for death in CRKP-infected patients.Results:The all-cause mortality rate of patients with CRKP bloodstream infection during hospitalization was 38%(19/50). The age ((66.89±18.13) vs. (55.06±14.39) years old, t=2.555, P=0.014), charlson's comorbidity index ((6.11±2.87) vs. (3.19±1.97), t=4.256, P<0.001) of the death group was higher than that of the survival group. The proportion of patients with chronic obstructive pulmonary disease (42.1%(8/19) vs. 3.2%(1/31), χ2=9.574, P=0.002), Charlson's comorbidity index ≥5 (68.4%(13/19) vs. 22.6%(7/31), χ2=10.314, P=0.001), septic shock (36.8%(7/19) vs. 6.5%(2/31), χ2=5.456, P=0.020), source of lung infection (36.8%(7/19) vs. 9.7%(3/31), χ2=3.868, P=0.049) was higher in death group than those in survival group. Kaplan-meier survival curve showed that the 30-day mortality of appropriate targeted treatment was lower than that of inappropriate targeted treatment ( χ2=8.138, P=0.004). Multivariate analysis showed that septic shock ( OR=56.363, 95% CI: 4.309-737.273, P=0.002) and charlson's comorbidity index ≥5 ( OR=18.605,95% CI: 1.813-190.896, P=0.014) were independent risk factors for mortality in patients with bloodstream CRKP infection. Conclusion:Appropriate targeted therapy can reduce 30-day mortality in patients with CRKP bloodstream infection. In order to reduce the risk of mortality, we should prevent the occurrence of septic shock and strengthen the diagnosis and treatment of patients with Chalson's comorbidity index ≥5.

Purpose: Reduced quality of life after cystectomy has made bladder preservation a popular research topic for muscle-invasive bladder cancer (MIBC). Previous research has indicated significant tumor downstaging after neoadjuvant chemotherapy (NAC). However, maximal transurethral resection of bladder tumor (TURBT) was performed before NAC to define the pathology, impacting the real evaluation of NAC. This research aimed to assess real NAC efficacy without interference from TURBT and apply combined modality therapies guided by NAC efficacy. Materials and Methods: Patients with cT2-4aN0M0 MIBC were confirmed by cystoscopic biopsy and imaging. NAC efficacy was assessed by imaging, urine cytology, and cystoscopy with multidisciplinary team discussion. Definite responders (≤ T1) underwent TURBT plus concurrent chemoradiotherapy. Incomplete responders underwent radical cystectomy or partial cystectomy if feasible. The primary endpoint was the bladder preservation rate. Results: Fifty-nine patients were enrolled, and the median age was 63 years. Patients with cT3-4 accounted for 75%. The median number of NAC cycles was three. Definite responders were 52.5%. The complete response (CR) was 10.2%, and 59.3% of patients received bladder-sparing treatments. With a median follow-up of 44.6 months, the 3-year overall survival (OS) was 72.8%. Three-year OS and relapse-free survival were 88.4% and 60.0% in the bladder-sparing group but only 74.3% and 37.5% in the cystectomy group. The evaluations of preserved bladder function were satisfactory. Conclusion After stratifying MIBC patients by NAC efficacy, definite responders achieved a satisfactory bladder-sparing rate, prognosis, and bladder function. The CR rate reflected the real NAC efficacy for MIBC. This therapy is worth verifying through multicenter research.

Objective To investigate the dose of docetaxel appropriate for patients with metastatic castration-resistant prostate cancer and its affects to the prognosis.Methods A retrospective analysis was performed on the clinical data of 75 patients with metastatic castration-resistant prostate cancer admitted from March 2010 to July 2016 who received docetaxel combined with prednisone chemotherapy.The patients were divided into the low-dose group (n =43,docetaxel ＜ 65 mg/m2),the middle-dose group (n =21,docetaxel 65-70 mg/m2) and the high-dose group (n =11,docetaxel ＞ 70 mg/m2).The median age in the low-dose group,middle-dose group and high-dose group was 67 (53-80),66 (56-78) and 61 (47-76) years old,respectively.Among 75 patients with bone metastasis,2 patients had no evidence of bone metastasis in the low-dose group.The lymph node metastasis was found in 26,13 and 6 cases in each group,respectively.And visceral and other metastasis were founded in 11,4 and 2 cases,respectively.The Gleason score in the low-dose group was≤7 points in 15 cases,≥8 points in 22 cases and no score in 6 cases.The Gleason score inthe middle-dose group was ≤7 points in 4 cases,≥8 points in 13 cases and no score in 4 cases.The Gleason score in the high-dose group was ≤7 points in 3 cases,≥8 points in 5 cases and no score in 3 cases.The number of patients with pain in the low-dose group,middle-dose group and high-dose group was 36,12 and 9,respectively,there were no significant differences in the above indicators (P ＞ 0.05),except age,which showed relatively more aged patients in the low-dose group,(P =0.045).Kaplan-Meier method was used to compare the overall survival (OS),progression-free survival (PFS) and the incidence of ≥CTCAE-4 grade 3 adverse reactions between the two groups.The Cox regression model was adopted to analyzed the factors that might affect patient prognosis,including the effective time of first-line endocrine therapy,hemoglobin level,ECOG score,pain score,number of cycles of chemotherapy,age,dose of docetaxel and alkaline phosphatase (ALP).Kaplan-Meier method was used to analyze the effect of dose of docetaxel on the prognosis,and log-rank method was used to test the significance of the results.Results The median OS was respectively 24.1,18.5 and 23.5 months in the low-dose group,middle-dose group and high-dose group,respectively.The median PFS was 5.3 months in all three groups,which didn't show statistically significant differences.The incidence of grade 3/4 adverse reactions in the low-dose group,middle-dose group and high-dose group was 15 cases (34.9％),8 cases (38.1％) and 5 cases (45.5％) respectively.It showed an increasing trend,but no statistically significant difference.The single factors related to OS mainly include the effective time of first-line endocrine therapy,hemoglobin level,ECOG score,pain score,number of cycles of chemotherapy,there was no significant correlation with age,docetaxel dose,ALP and PSA value.Conclusions It is common to receive lower doses of docetaxel in clinical practice for patients with metastatic castration-resistant prostate cancer in China.The efficacy of low-dose docetaxel is similar to that of high doses (standard dosage).There was no significant correlation between the OS and the actual dose of docetaxel in the tolerable range.

Objective To evaluate the efficacy and safety of the modified docetaxel plus prednisone scheme for the metastatic castration resistant prostate cancer patients who got poor tolerance to chemotherapy.Method The clinical data of 50 metastatic castration resistant prostate cancer who received docetaxel + prednisone chemotherapy from March 2010 to October 2015 were analyzed retrospectively.23 cases received the modified DP regimen (modified group),27 cases received the standard DP regimen (standard group).The median age of the modified group and the standard group were 69 years (47-80 years) and 63 years (52-77 years) (P =0.005).There were 19 and 24 cases with pain in modified group and standard group respectively;10 and 19 cases with lymph node metastasis respectively;3 and 4 cases of visceral metastasis respectively;all of the 50 patients were complicated with bone metastasis.For the pathological Gleason score,there were 7 cases scored ≤7 points,13 cases scored ≥ 8 points and 3 cases unscored in the modified group;7 cases scored ≤7 points,15 cases scored ≥8 points and 5 cases unscored in standard group.There was no significant difference of the pain,metastasis,and Gleason score between the two groups (P ＞ 0.05).Progression free survival (PFS),overall survival (OS)and adverse events were analyzed using Kaplan-Meier curves,and the differences were assessed using the log-rank test.Results In the modified group and standard group,the median follow-up times were 11.0 months and 14.0 months respectively,the median chemotherapy cycles were 4.5 cycles and 5.0 cycles respectively;OS were 18.0 months and 27.5 months respectively (P =0.746).The PFS of the two groups were 6.0 months and 5.2 months,respectively (P =0.822).The PSA response were 13 cases and 17 cases in the modified group and standard group respectively (P =0.615),and the pain response were 8 cases and 7 cases (P =0.927),grade 3 to 4 adverse events were 3 cases and 14 cases (P =0.003).The main adverse events were blood toxicity,neutrophils,gastrointestinal reaction,edema,fatigue and oral mucositis etc.Conclusions Compared with the standard DP scheme,the modified DP scheme had no significant difference in OS,PFS,pain response rate and PSA response rate,while the incidence of grade 3 to 4 adverse events was significantly reduced.Modified DP scheme may be a better choice for patients with metastatic castration resistant prostate cancer who get poor tolerance to chemotherapy.

Objective To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein (GFP), and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7 cells. Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells. Different concentrations (0.5 and 1.0 mmol · L-1 ) of isopropyl-β-D-thiogalactopyranoside (IPTG ) and cell culture temperatures (22℃ and 37℃)were used to optimize the protein expression.The Tat-GFP proteins in supernatant were purified using Ni-IDA resins. Western blotting analysis was used to identify the Tat-GFP protein, and confocal laser scanning microscope (CLSM ) was used to examine the cell penetration of Tat-GFP protein. Results There was no significant difference in the Tat-GFP protein production induced by 0.5 and 1.0 mmol·L-1 IPTG;however,the low temperature (22℃)-induced BL21 cells expressed more Tat-GFP proteins than that at 37℃ induction.The Western blotting analysis results showed that GFP antibody could specifically recognize the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7 cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL2 1 cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.

Objective To explore the effect of acupuncture on trunk control ability and balance function in patients with spinal cord injury above lumbar. Methods 40 patients with spinal cord injury were randomly divided into observation group and control group with 20 cases in each group. The control group received limb joint training method, the observation group received acupuncture in addition. Fugl-Meyer Assessment and Flex Comp Infiniti EMG system were used to evaluate the balance function and trunk muscle strength before and 8 weeks after treatment. Results After treatment, the balance function and trunk control ability improved in both groups (P<0.01), and were better in the observation group than in the control group (P<0.05). Conclusion Limb linkage combined with acupuncture can further improve the balance function and trunk control ability in patients with spinal cord injury above lumbar.