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A gap is found between the existing quality of care,and social expectations and the PDCA model.Peking University People's Hospital applied the quality control circle in building a supervision platform for medical quality and sustained improvement of civilized services.This study demonstrated the efforts in this regard made by the hospital,and the major outcomes of the platform in its 6-year practice in identifying service defects,optimizing service processes and sustained improvement of medical services.In addition,it also shared insights on enhanced real-time supervision,standardized management,and long-term mechanisms of the hospital.
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Objective To investigate the proliferation, immune phenotype and cytotoxicity on different cell lines of cytokine-induced killer (CIK) cells collected from healthy donors. Methods Peripheral blood mononuclear cells (PBMC) from healthy donors were induced to become CIK cells by adding cytokines including rhIL-2, rhIFN-γand CD3 McAb. The proliferation of CIK cells was tested by blood cell recording board. The CIK cells were analyzed on different time points by FACS. The cytotoxicity of CIK cells against different tumor cell lines, such as K562, BJAB, A549, MCF-7 and HepG2, was detected by MTT assays on day 13. Results CIK cells quickly proliferated from day 5, and expanded by 182-fold after 20-day culture. The immunophenotypes of CD3+, CD3+CD8+and CD3+CD56+were (97.83±1.03)%, (77.12±1.60)%and (27.58± 2.02)%. The percentages of CD3+, CD3+CD8+and CD3+CD56+increased noticeably (P＜0.01). According to the effector-tar-get ratio of 40∶1, the activity of CIK cells against tumor cells K562, BJAB, A549, MCF-7 and HepG2 were (88.89±7.22)%, (75.42±9.52)%, (63.19±5.67)%, (43.53±5.67)%and (42.63±7.69)%. The experiments showed that CIK cells possessed high-er antitumor cytotoxic activity. Conclusion CIK cells can be largely capacity cultured by adding cytokines in vitro. CIK cells were a highly efficient cytotoxic cell against tumors, and had clinical application potentials.
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Objective To search for a scientific and feasible system for performance appraisal of hospital management staff, and enhance the level of hospital management. Methods The 360-degree evaluation and improved performance report evaluation are called into play to examine such staff. Examiners include the superiors, peers (heads of clinical and technical departments and those of administrative departments), inferiors and others (representatives of the faculty congress and staff congress). Results Outcomes of the two evaluation methods found 8 of the top ten as overlapping, and 8 of the bottom ten as overlapping as well. 80% overlapping between the two prove the results as highly similar, and a relativity analysis found the general results of the two methods as highly consistent. Conclusion United applic of the two methods can effectively examine the management staff and improve their management capabilities.
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Objective:To prepare and characterize specific and discrepant mouse hybridoma antibodies on membrane of HL60 and HL60/ADR cell lines.Methods:BALB/c mice were immunized by subtractive immunization induced Cp(Cyclophosphamide).McAbs were prepared by hybridoma technique,screened and detected by FACS and LSCM.Results:51 candidates and discrepant antibodies were found,and one of them (5F6) was purified and identified.Conclusion:Combination of SI with discrepant screening method should facilitate the preparing and identifying discrepant McAbs for identifying antibodies that can distinguish the differences in proteins expressed in HL60 and HL60/ADR,which is a significative and potential method in the research and target therapy associated drug-resistance.
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We constructed and expressed an anti-CD3/anti-Pgp (P-glycoprotein) diabody previously. However, the two chains of diabody are associated non-covalently, resulting in being capable of dissociating. The aim of this study is to enhance the stability of the diabody. We introduced cysteine residues into the CD3 or Pgp V-domain to covalently lock the two chains together. The disulphide crosslinked diabody were expressed by Escherichia coli (E. coli) 16C9 and purified by a cation exchange column and an anti-Etag affinity chromatography. The purified proteins were verified through SDS-PAGE. Flow cytometry (FCM) was used to analyse the binding properties, competitive binding capacity and stability in vitro. The dsPpg-diabody failed to form disulphide bond properly. The designed disulphide bridge between the different chains of dsCD3-diabody was formed correctly. FCM demonstrated the dsCD3-diabody has specific antigen binding activity, the same binding activity and competitive binding activity as its parent diabody. The dsCD3-diabody retained the full activity even after 72 h incubation at 37 degrees C in human serum, in contrast, the parent diabody began to lose activity after only 1 h and lose all its activity 24 hours later. The induced disulphide bond in the CD3 V-domain effectively enhanced the stability of anti-CD3/anti-Pgp diabody. The method of stabilizing a diabody by introducing a disulphide bond into is practical.
Subject(s)
Humans , ATP Binding Cassette Transporter, Subfamily B , Allergy and Immunology , Antibodies, Bispecific , Chemistry , Genetics , Allergy and Immunology , Binding, Competitive , CD3 Complex , Allergy and Immunology , Cell Line , Disulfides , Chemistry , Drug Stability , Escherichia coli , Genetics , MetabolismABSTRACT
Objective To evaluate the specificities and sensitivities of neuron-assoeiated(Neu-A) autoantibodies in the diagnosis of neuropsychiatric systemic lupus erythematosus(NPSLE)and explore its role in the pathogenesis of NPSLE.Methods The ncuro-associated autoantibodies including anti-neuroblastoma cell,anti-neuroglioma cell,and anti-brain,anti-spinal antibodies were tested using neuroblastoma cell line SK-N-SH.ueuroglioma cell line U251 or cerebrum and spinal meduHa of mice C57BL/10 as substrates respec tively,Serums of 121 SLE patients fincluding 36 NPSLE patients and 85 systemic lupus erythematosus (SLE)patients without CNS involvement),34 rheumatoid arthritis(RA)patients and 34 healthy controls were tested by indirect immunofluorescence(IIF)assay.Cerebrospinal fluid of 24 NPSLE patients and 22 CNS dis eases controls were also tested.Results The positive rates of anti-neuroblastoma cell antibodies,anti-brain and anti-spinal antibodies in serums of SLE patients were 17.4%,25.6%and 29.8%,respectively.Only 2.9%, 5.9%,0 in 34 RA patients and 0,2.9%,0 in health controls were positive(P＜0.05 or P＜0.01).The specificity of the three antibodies in SLE was 98.5%.95.6%and 100%respectively.Anti-neuroblastoma cell antibodies, anti-brain and anti-spinal antibodies were closely associated with SLEDAI score.The NPSLE patients with positive anti-neuroblastoma cell antibodies,anti-brain or anti-spinal antibodies had frequent seizure atlacks, headache,acute confusion state and mood disorders.There was a higher prevalence of anti-neuroblastoma cell.anti-brain and anti-spinal antibodies in NPSLE than those in SLE patients.29.2%anti-brain and anti- spinal antibodies were detected in the eerebrospinal fluid of NPSLE patients.which were significantly higher than those of disease control group(P＜0.05 or P＜0.01).The specificities of these two antibodies jn NPSLE were 100%and 95.5%respectively.Conclusion Anti-neumhlastoma cell antibody.anti-brain and anti spinal antibodies are valuable markers for the diagnosis of SLE.They are closely associated with the disease activity of SLE and neuropsychiatric symptoms.Anti-spinal antibody may be a sensitive and specific marker for the diagnosis of NPSLE while anti-brain and anti-spinal antibodies in the cerebrospinal fluid are more specifie for the diagnosis of NPSLE.
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Aim To determine whether membrane cytokeratin 8(CK8 )and BCRP expression cooperatively contributed to multidrug resistance(MDR)in MCF-7/MX cells.Methods MCF-7/MX cells were transfected with specific anti CK8-siRNAs and anti BCRP-siRNAs via LipofectAMINE2000.The expression of CK8 and BCRP was determined using Western blot,and membrane staining was observed by laser confocal microscopy.Sensitivity to chemical drugs was examined by Sulforhodamine B method.Results The expression levels of cell surface CK8 and BCRP were obviously reduced by siRNAs,and inhibition of CK8 and BCRP expression could effectively restore the sensitivity to drugs and reverse MDR phenotype of MCF-7/MX cells.Conclusions CK8 together with BCRP may play significant roles in conferring the multifactorial MDR phenotype of MCF-7/MX cells,but may act independently via potentially different mechanisms.Combinational approaches that target multiple drug-resistance-related molecules/pathways in cancer cells may represent more efficacious strategies to overcome MDR.
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Aim To look for novel small-molecule inhibitors of CDK9 through structure-based virtual screening and biological activity determination.Methods Homology modeling of CDK9 was based on the 3-D structure of other cyclin-dependent kinase family members,and then virtual screening by DOCK(molecular docking)of database of small molecule was carried on.MTT method was used in inhibition of tumor cell growth in vitro,while Western blot was used for further study of molecular mechanisms.Results From the top 1000 compounds with the best DOCK energy score,27 compounds were selected for biological assay based on the diversity of chemical structure and functional group.12 of 27 selected compounds showed significantly inhibition activity on tumor cell proliferation,and only one compound in 12 with half-maximum inhibition concentration(IC50)values less than 20 ?mol?L-1 named C-21 was selected for further molecular mechanism study.The western blotting data showed C-21 compound could effectively inhibit CDK9 from phosphorylating large subunit C-terminal of RNA polymerase Ⅱ in a dose-dependent manner.Conclusions Through homology modeling,virtual screening by computer,determination of biological activity and experimental studies of molecular mechanism,a new promising lead compound targeted for CDK9 was found and confirmed.