Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

Objective:To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods:Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the V H from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results:Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×10 11 and 1.54×10 11, respectively. The titers for two antibody libraries were 6.04×10 13 CFU/ml and 3.50×10 13 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion:This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology.

The design of the salary structure in public hospitals is an important part of the salary system reform.The salary structure is in a dynamic evolutionary process as the reform deepens,and at this stage,it is gradually shifting towards a development direction that emphasizes incentive-based salary performance.Continuous optimization of the salary structure is key to exploring the accurate measurement of doctors' value and the sustainable development of the hospital's public welfare attributes.Through literature research and comparative analysis,it presented a tabular presentation of the four incentive compensation models formed so far in the dynamic optimization of China's public hospitals after the new healthcare reform,and to conduct a comparative analysis of their respective advantages and disadvantages,in order to provide reference and information for the continuous optimization of the pay structure of public hospitals.

The design of the salary structure in public hospitals is an important part of the salary system reform.The salary structure is in a dynamic evolutionary process as the reform deepens,and at this stage,it is gradually shifting towards a development direction that emphasizes incentive-based salary performance.Continuous optimization of the salary structure is key to exploring the accurate measurement of doctors' value and the sustainable development of the hospital's public welfare attributes.Through literature research and comparative analysis,it presented a tabular presentation of the four incentive compensation models formed so far in the dynamic optimization of China's public hospitals after the new healthcare reform,and to conduct a comparative analysis of their respective advantages and disadvantages,in order to provide reference and information for the continuous optimization of the pay structure of public hospitals.

DRG payment reform is an important means to control the unreasonable growth of medical expenses, improve the quality of medical services and achieve a win-win situation among three sides of hospitals, medical insurance and patients. This study adopted the Smith policy implementation process model to analyze the difficulties in the DRG policy implementation process from four aspects(idealized policies, policy implementation institutions, target groups, and policy environment), including the deviation between policy connotations and actual needs; the interest objectives of all parties were not completely aligned, the target group lacked a sense of identity, and the social impact and technological support needed to be improved. It was suggested that optimization should be carried out from four dimensions: policy supply coordination and precision, performance evaluation and personnel literacy, target group cognitive level and participation willingness, and policy implementation environment and atmosphere, in order to synergistically promote the effective implementation of DRG policies.

ObjectiveTo study the effect of Bushen Huoxuetang on the apoptosis and the expression of B-cell lymphoma （Bcl-2）-associated X protein （Bax）/ Bcl-2 and cleaved cysteine-containing aspartate proteolytic enzyme-3 （cleaved Caspase-3） in the nude mouse model of bone metastasis of breast cancer， and explore the mechanism of Bushen Huoxuetang in inhibiting bone destruction. MethodThirty BALB/c female nude mice were randomly assigned into blank group （n=6） and model group （n=24）. The suspension of 4T1 breast cancer cells was injected into the tibia of mouse right lower limb to establish model of bone metastasis of breast cancer. The successfully modeled nude mice were randomly assigned into model group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group， with 6 mice in each group. Bushen Huoxuetang was administrated at a dose of 36.67 g·kg^-1， once a day， and zoledronic acid was administrated by subcutaneous injection at a dose of 100 μg·kg^-1， twice a week. The combined drug group was administrated with the same doses of Bushen Huoxuetang group by gavage and zoledronic acid by subcutaneous injection. The mice in the blank group and the model group were administrated with the same volume of distilled water by gavage for 14 days. On the next day at the end of drug administration， the mice were sacrificed by cervical dislocation. The general situation and weight changes of the mice were examined. The right lower limb was collected， and X-ray scanning and hematoxylin-eosin （HE） staining methods were used for observation of pathological changes in the bone. The terminal deoxynucleotidyl transferase dUTP nick-end labeling （TUNEL） was employed to detect the apoptosis of bone tissue in nude mice， and Western blot to determine the expression of Bax/Bcl-2 and cleaved Caspase-3 in the bone tissue. ResultCompared with the blank group， the modeling reduced the body weight （P<0.01） and increased the right lower limb weight of the nude mice （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination increased the body weight （P<0.01） and decreased the right lower limb weight （P<0.01）. Compared with the blank group， the other groups showed obvious tumor cell atypia， deep nuclear staining， and clear bone metastasis， and the model group showed obvious osteolytic damage in right lower limb and loss of proximal tibia and knee joint. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination reduced the osteolytic lesions in the right lower limb and recovered part of the bone structure， demonstrating an inhibitory effect on bone destruction. The TUNEL assay showed that the model group had lower apoptosis rate of bone metastatic tumor cells than the blank group， Bushen Huoxuetang group， zoledronic acid group， and combined drug group （P<0.01）. Compared with the blank group， the modeling down-regulated the expression of Bax and cleaved Caspase-3 （P<0.01） and up-regulated the expression of Bcl-2 （P<0.01）. Compared with the model group， Bushen Huoxuetang， zoledronic acid， and their combination up-regulated the expression of Bax （P<0.01） and cleaved Caspase-3 （P<0.05， P<0.01） and down-regulated the expression of Bcl-2 （P<0.05， P<0.01）. ConclusionBushen Huoxuetang may inhibit bone destruction in the nude mouse model of bone metastasis of breast cancer by up-regulating the expression of Bax， down-regulating the expression of Bcl-2， activating cleaved Caspase-3， and further inducing apoptosis.

Objective To analyze the expressions of thymidine kinase 1 (TK1) and nuclear-associated antigen Ki-67 in triple negative breast cancer (TNBC) and their clinical significances.Methods One hundred and twenty tumor tissue sections of patients with breast cancer who were performed breast conservation treatment or modified mastectomy in the Second Affiliated Hospital of Qingdao University Medical College from June 2009 to December 2010 were collected,and there were 60 cases with TNBC and 60 cases with non-TNBC.The expressions of TK1 and Ki-67 in different breast tissues were detected by immunohistochemistry.The relationships between the expression status and clinicopathologic features were analyzed.Results The positive expression rates of TK1 in TNBC and non-TNBC were 83.33％ and 51.67％ respectively,with a significant difference (x2 =13.713,P =0.000).The positive expression rates of Ki-67 expression in TNBC and nonTNBC were 68.33％ and 31.67％ respectively,with a significant difference (x2=16.133,P =0.000).In TNBC,the expression of TK1 was related to histological staging (x2 =6.125,P =0.013),but it was not related to onset age (x2 =0.809,P =0.369),menopausal stutas (x2 =1.615,P =0.204),tumor size (x2 =0.054,P =0.816) and lymphatic metastasis (x2 =0.672,P =0.412).In TNBC,the expression of Ki-67 was related to histological staging (x2 =13.145,P =0.000) and lymphatic metastasis (x2 =6.182,P =0.013),but it was not related to menopausal stutas (x2 =1.018,P =0.313),onset age (x2 =2.377,P =0.123) and tumor size (x2 =2.401,P =0.121).The expression of TK1 was positively correlated with that of Ki-67 (r =0.369,P =0.023).The results of survival analysis showed that the disease-free survival rates of 5-year were 28.20％ and 66.70％ in the TK1 positive group and TK1 negative group,and the disease-free survival rates of 5-year were 24.30％ and 64.30％ in the Ki-67 positive group and Ki-67 negative group,with significant differences (x2 =4.194,P=0.041;x2 =4.540,P =0.033).Conclusion TK1 and Ki-67 are highly expressed in TNBC,and their expressions are correlated with histological staging and survival,which are expected to become prognostic indicators.

OBJECTIVETo analyze the 3 dimensional-fat suppression-spoiled gradient-recalled acquisition (3D-FS-SPGR) sequence in the diagnosis of knee articular cartilage injury.

METHODSA total of 56 knee osteoarthritis patients (26 males, 30 females, ages 52-73 years, mean 61.8 years) treated in Department of Orthopedics, Chinese People's Liberation Army General Hospital between June 2013 and May 2014 were involved in this study. All patients underwent knee MRI, plus 3D-FS-SPGR sequence, arthroscopic exploration, and in contrast to the results of MRI results analysis, evaluation 3D-FS-SPGR and conventional sequence of cartilage damage consistent with the arthroscopic accuracy.

RESULTSDivided 56 knee joints into 336 cartilage articular surface, included 55.1% normal articular surface, 21.4% early osteoarthritis and 23.5% advanced osteoarthritis. The accordance of 3D-FS-SPGR sequence grading and arthroscopic was 90.2%. The sensitivity of 3D-FS-SPGR sequence was 93.1%, specificity was 98.3%, and Kappa value was 0.849. The sensitivity of T2WI sequence was 84.4%, specificity was 96.9%, and the Kappa value was 0.671.

CONCLUSIONFor unicompartment osteoarthritis , MRI 3D-FS-SPGR sequence is effective in sensitivity and specificity of cartilage damage.

Aged ; Arthroplasty ; Arthroscopy ; Cartilage, Articular ; Female ; Humans ; Knee Injuries ; diagnosis ; Knee Joint ; physiopathology ; surgery ; Magnetic Resonance Imaging ; methods ; Male ; Middle Aged ; Orthopedic Procedures ; Osteoarthritis, Knee ; surgery ; Preoperative Care ; Sensitivity and Specificity

BACKGROUNDPatellofemoral osteoarthritis commonly occurs in older people, often resulting in anterior knee pain and severely reduced quality of life. The aim was to examine the effectiveness of arthroscopic patelloplasty and circumpatellar denervation for the treatment of patellofemoral osteoarthritis (PFOA).

METHODSA total of 156 PFOA patients (62 males, 94 females; ages 45-81 years, mean 66 years) treated in our department between September 2012 and March 2013 were involved in this study. Clinical manifestations included recurrent swelling and pain in the knee joint and aggravated pain upon ascending/descending stairs, squatting down, or standing up. PFOA was treated with arthroscopic patelloplasty and circumpatellar denervation. The therapeutic effects before and after surgery were statistically evaluated using Lysholm and Kujala scores. The therapeutic effects were graded by classification of the degree of cartilage defect.

RESULTSA total of 149 cases were successfully followed up for 14.8 months, on average. The incisions healed well, and no complications occurred. After surgery, the average Lysholm score improved from 73.29 to 80.93, and the average Kujala score improved from 68.34 to 76.48. This procedure was highly effective for patients with cartilage defects I-III but not for patients with cartilage defect IV.

CONCLUSIONSFor PFOA patients, this procedure is effective for significantly relieving anterior knee pain, improving knee joint function and quality of life, and deferring arthritic progression.

Aged ; Aged, 80 and over ; Cartilage, Articular ; innervation ; surgery ; Denervation ; methods ; Humans ; Knee Joint ; innervation ; surgery ; Middle Aged ; Osteoarthritis, Knee ; surgery ; Patellofemoral Joint ; innervation ; surgery ; Quality of Life

Objective To identify the factors related to radiation pneumonia (PR) in lung cancer treated with intensity modulated radiotherapy (IMRT).Methods Data from 163 lung cancer patients treated with IMRT were analyzed with clinical factors and physical parameters related to the dose-volume histogram.The patients were followed for 6 months after radiotherapy.The relationship between survival status and PR was analyzed.Results The incidence rate of over grade 2 patients was 28.22 ％ (46/163).Univariate analysis revealed a significant relationship between many parameters associated with such as the site of lobe (P =0.033),COPD (P =0.020),chemotherapy (P =0.020) and prophylactic using of glucocorticoid and antibiotic (P =0.025).Multilogistic regression analysis showed that V20 in the contralateral site,the V5 of the all lungs and PTVV were independent factors.Conclusion The RP is associated with multiple factors.Individualized treatment plans should be made according to the specific circumstances of patients.