Objective:To clarify the effect of ubiquitination hepatitis B virus core antigen (Ub-HBcAg) on dendritic cells (DC) autophagy, and to explore the mechanism of autophagy in enhancing DC antigen presentation and inducing hepatitis B virus-specific cytotoxic T lymphocyte (CTL) responses.Methods:Ub-HBcAg lentiviral vector (LV-Ub-HBcAg), lentiviral vector-hepatitis B virus core antigen (LV-HBcAg) and no-load plasmid LV (LV) were constructed and packaged. DC2.4 cells were divided into LV-Ub-HBcAg group, LV-HBcAg group and LV group. The blank control group (NC group) was also set. The protein expression of autophagy-related protein P62, microtubule associated protein 1 light chain 3 beta (LC3B), autophagy related 5(ATG5) and Beclin-1 were detected by Western blotting. The expressions of co-stimulatory molecules such as CD86, CD80 and major histocompatibility complex (MHC)-Ⅱ were detected by flow cytometry. Cell counting kit-8 (CCK-8) method was used to detect T lymphocytes proliferation. The non-radioactive lactic acid dehydrogenase (LDH) release method was applied to detect the killing ability of CTL. Statistical analysis was conducted by independent sample t test. Results:The relative protein expressions of LC3B-Ⅱ/LC3B-Ⅰ, Beclin-1 and ATG5 in NC group were 0.445±0.076, 0.522±0.026 and 0.761±0.038, respectively, which were all lower than those in LV-Ub-HBcAg group (0.926±0.021, 0.919±0.016 and 1.451±0.028, respectively). The relative protein expression of P62 in the NC group was higher than that in LV-Ub-HBcAg group ((1.875±0.016) vs (0.647±0.121)). The differences were all statistically significant ( t=6.102, 9.842, 17.490 and 10.590, respectively, all P<0.01). The expressions of CD86 (75.51%), CD80 (83.35%), MHC-Ⅱ (66.66%) in the LV-Ub-HBcAg group were high, and those in the NC group were 8.03%, 7.49%, 0.04%, respectively. The specific CTL killing rate ((65.310±2.091)%) of the LV-Ub-HBcAg group was significantly higher than both NC group ((14.400±0.497)%) and LV-HBcAg group ((54.870±1.443)%), and the differences were both statistically significant ( t=23.690 and 4.111, respectively, both P<0.05). Conclusion:Ub-HBcAg promotes the DC autophagy, up-regulates the expressions of costimulatory molecules on cell surface of DC to induce the maturation and activation, and then stimulates T lymphocyte to induce a stronger specific CTL response under the effort of ubiquitination.

Objective To observe the effects of cytoplasmic transduction peptide (CTP)-HBcAg18-27-Tapasin induced murine bone marrow-derived dendritic cell (DC) maturation on T lymphocyte proliferation in vitro,Methods Bone marrow derived DC isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleutin (IL)-4 for 5 days followed by lipopolysaccharide added to induce DC maturation.10 μg/L CTP-HBcAg18-27-Tapasin,50 μg/L CTP-HBcAg18-27-Tapasin,10 μg/L CTP-HBcAg18-27 or RPMI-1640 were added into culture medium to induce DC maturation.DC phenotypes were analyzed by flow cytometry.The level of IL-12p70 in the supernatant was detected by enzyme linked immunosorbent assay.The proliferation of.T lymphocytes was performed by using cell counting kit-8 and intracellular cytokine of proliferative T cells were analyzed by flow cytometry.The means among groups were compared using one-way ANOVA and those between two groups were compared by least significant difference test.Results DC were cultured and induced successfully.The molecules on DC surface,such as CD80,CD86 and major histocompatibility antigen-Ⅰ were upregulated by CTP-HBcAg18-27-Tapasin.IL-12p70 level induced by 50 μg/L CTP-HBcAg18-27-Tapasin was (61.12±10.25) pg/mL,which was higher than those induced by 10 μg/L CTP-HBcAg18-27-Tapasin (50.43±10.42) pg/mL,10μg/L CTP-HBcAg18-27 (40.17±8.54) pg/mL and medium control (30.51±8.03) pg/mL (F=15.85,P=0.030 and 0.037).The proliferation of T lymphocytes induced by CTP- HBcAg18-27 -Tapasin was higher than control groups.The amounts of cytotoxic T lymphocyte (CTL) induced by 50 μg/L CTP-HBcAg18-27-Tapasin [(2.05±0.41) ％] and 10 μg/L CTP-HBcAg18-27-Tapasin [(1.06 ±0.10 )％] were both significantly higher than the 10 μg/L CTP-HBcAg18-27 group [(0.45±0.11)％] and medium group [(0.09±0.02)％,F=60.22,P=0.003].Conclusions CTP HBcAg18- 27 Tapasin could promote the differentiation and maturation of DC,and enhance the ability of DC stimulating T lymphocytes proliferation and increase CTL expression effectively.

Objective To observe the changes of calcitonin gene-related peptide(CGRP) -immunoreactive positive nerve fibers innervation in bone tissue of femoral heads during the pathological process of early steroid-induced avascular necrosis of the femoral head (SANFH), and explore its significance.Methpathological group of SANFH was induced.Immunohistochemical technique was used and the changes of CGRP-immunoreactive positive nerve fibers innervation in weight bearing area of the femoral heads during the pathological process of early SANFH were observed.Result The number of CGRP-immunoreactive nerves increased first and then decreased ( Peaked at 6 weeks, 10.28 ± 0.66 ), but it was more than that ofnormal control group.There was significant difference between two groups ( P ＜ 0.01 ).Conclusion There were changes in the distribution of CGRP-immunoreactive positive nerves fiber during the process of bone repair after SANFH.CGRP-immunoreactive positive nerves fiber might take part in the process of bone repair in SANFH.

AIM: To Screen and identify human single-chain variable fragment (ScFv) specific to hepatitis B virus core protein and determine its gene sequence. METHODS: The recombinant phages were panned by HBcAg coated in a 96-pore plate and 48 clones were identified specific to HBc after three rounds of panning. The specificity of ScFv from the positive clone was determined by ELISA. Then, the soluble ScFv was expressed in E.coli. HB2151 and secreted in the supernatant. Subsequently, SDS-PAGE and dot blot were performed to identify the ScFv in the supernatant and cell lysate. The gene of ScFv specific to hepatitis B virus core protein was sequenced. RESULTS: The ScFv screened from phage antibodies has a specific combination character with hepatitis B virus core antigen. Soluble ScFv was confirmed to express in E.coli. HB2151 and secrete in the supernatant. The sequence of ScFv gene conformed to that of heavy chain and kappa chain of human immunoglubulin. CONCLUSION: Human ScFv specific to hepatitis B virus core protein has been identified by means of the phage display technology, and its gene sequence has been determined.

Objective: To study the influencing factors of fracture nonunion after intramedullary nailing for subtrochanteric fracture and construct a risk assessment model. Methods: A retrospective analysis was performed on 251 patients with intramedullary nail fractures of the femoral subtrochanteric fracture from February 2006 to January 2018. According to the different treatment time, the 251 patients included in this study were divided into the modeling group and the verification group. In the modeling group, postoperative fracture nonunion rate, general data, fracture related factors, surgical reduction related factors, mechanical and biological factors were calculated, and the influencing factors of fracture nonunion were screened by univariate analysis. Indicators with statistical differences in univariate analysis were analyzed using Logistic regression model for multivariate analysis to build the risk assessment model. The influencing factors were re-evaluated through the verification group, and the differentiation and calibration of the model were evaluated. Results: Fracture nonunion occurred in 34 of 149 patients in the modeling group. Among the 13 potential influencing factors, univariate analysis and logistic regression analysis showed that postoperative hip varus, intramedullary nail fixation failure and complete open reduction were the risk factors of fracture nonunion. Postoperative reduction of medial cortex was a protective factor for fracture nonunion, and a regression equation was established. Based on the logistic regression model, the Nomogram diagram was drawn. In the verification group, fracture nonunion occurred in 24 of 149 patients. The area under the ROC curve was AUC=0.883>0.7, indicating that there was a moderate differentiation to evaluate the occurrence of fracture nonunion after operation. The goodness of fit test: the H-L test (χ²=2.921, P=0.712) showed that the model had a good calibration. Conclusion The risk factors of fracture nonunion were hip varus, failure of intramedullary nail fixation and complete open reduction after intramedullary nailing of subtrochanteric fracture, and postoperative reduction of medial cortex was the protective factor. The risk assessment model has moderate differentiation and good calibration, which can provide reference for the risk assessment of fracture nonunion after subtrochanteric fracture operation.

OBJECTIVETo investigate the effect of protein transduction domain-hepatitis B virus core antigen (CTP-HBcAg18-27)-Tapasin fusion protein-induced specific cytotoxic T lymphocyte (CTL) response on hepatitis B virus (HBV) replication in HBV transgenic mice.

METHODSTwenty HBV-transgenic mice were randomly divided into two groups for a 3-week course of once weekly subcutaneous immunizations with either CTP-HBcAg18-27-Tapasin fusion protein or CTP-HBcAg18-27. Mice administered isotonic saline served as blank controls. Expressions of cytokines in splenocytes were analyzed by flow cytometry. Serum levels of hepatitis B surface antigen (HBsAg) and HBV DNA were determined by microparticle enzyme immunoassay and real-time fluorescent PCR assay, respectively. Expression of HBsAg in hepatic tissues was detected by immunohistochemistry.

RESULTSImmunization with 100 mug of CTP-HBcAg18-27-Tapasin fusion protein led to a significant increase in proportions of CTLs in spleen (2.70%+/-0.20% vs. 50 mug of CTP-HBcAg18-27-Tapasin: 1.66%+/-0.53%, 50 mug of CTP-HBcAg18-27: 1.26%+/-0.56%, and blank controls: 0.75%+/-0.71%; F = 741.45, P = 0.000) and up-regulation of inflammatory cells in hepatic tissue. In addition, both immunizations of CTP-HBcAg18-27-Tapasin led to significant decreases in serum HBsAg and HBV DNA levels compared to those in the CTP-HBcAg18-27 group.

CONCLUSIONHBV-related modification of the expression of the molecular chaperone Tapasin may affect its interaction with intracellular antigen peptides, thereby leading to increases the number of specific CTLs in the spleen, decreases in serum HBsAg and HBV DNA levels, and down-regulation of HBsAg expression in hepatic tissue. These results obtained in HBV-transgenic mice suggest that the CTP-HBcAg18-27-Tapasin fusion protein has anti-HBV activity.

Animals ; DNA, Viral ; blood ; Female ; Hepatitis B ; immunology ; Hepatitis B Core Antigens ; genetics ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; physiology ; Male ; Membrane Transport Proteins ; genetics ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Recombinant Fusion Proteins ; genetics ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Virus Replication

Bladder cancer is a kind of urothelial cancer with high incidence and heterogeneity. From superficial bladder tumors to muscular invasive malignant tumors, due to their high-frequency recurrence and metastatic characteristics, they are inherently refractory. Although a comprehensive treatment with surgery as the main focus while chemotherapy, radiotherapy, and immunotherapy as a supplement has been formed, the limitations of chemotherapy regimens, the unpopularity of radiotherapy, and the low efficiency of immunotherapy, make clinical decision-making still difficult. Recently, a number of targeted therapies have emerged in bladder cancer and have shown good responses. Transient receptor potential (TRP) channel are novel therapeutic targets and current research hotspots in bladder cancer. This review discusses the anti-tumoral molecular mechanism of TRP channel in bladder cancer, its feasibility as a potential therapeutic target, and the prospects of drug combination to sensitize platinum-based chemotherapy.

Objectives:To explore the feasibility of anterior medial fenestration approach in situ reduction and fixation in the treatment of PipkinⅠ and Ⅱ femoral head fractures,and to explore the clinical effect of this operation.Methods:Hips of two anti-corrosion adult specimens treated with formalin were dissected, then anatomical structures and directional characteristics of anterior medial main muscles,ligaments,blood vessels and nerves were observed.The anterior medial fenestration approach was performed on bilateral hips of four fresh frozen specimens to determine pulling direction of stripped muscles and ligaments required during operation,and to observe and analyze vascular and nerve traction protection directions exposed in the approach.Determine extent of exposure to the approach and assess feasibility of this approach.The clinical data of 12 patients with Pipkin Ⅰ and Ⅱ femoral head fractures who underwent in situ reduction and fixation of anterior medial fenestration at Department of Orthopaedics,Affiliated Hospital of Chengdu University from February 2016 to April 2018 were retrospectively analyzed.There were 3 males and 9 females with an age of 48.5 years(range:37 to 59 years).There were 8 cases of Pipkin type Ⅰ and 4 cases of Pipkin type Ⅱ.The operation time,blood loss,fracture healing time,last Thompson-Epstein evaluation and Harris score were observed.Results:Anterior medial fenestration approach to expose the femoral head in 4 bilateral hips with a total of 8 sides of fresh frozen specimens.The upper boundary of observation fenestration was pubic body (anterior acetabulum),and the outer upper boundary was iliacus and psoas muscle.The lateral boundary was rectus femoris and femoral vessels,the lower boundary was transverse branch of the medial femoral circumflex artery and vein.The medial boundary was pubis muscle,short adductor muscle and long adductor muscle.Pubofemoral and iliofemoral ligament were seen in fenestration. Four quadrants in front of femoral head in fenestration can be seen after cutting switch capsule active hip joint. In 12 patients with femoral head fracture,the operation time was 107.5 minutes(range:90 to 135 minutes),and the intraoperative bleeding volume was 115.0 ml(range:85 to 150 ml).The patients were followed up for 18.6 months(range:12 to 28 months).The fracture healing time of 12 patients was 144.2 days(range:120 to 180 days).The curative effect was evaluated according to Thompson-Epstein standard at the last follow-up:excellent in 6 cases,good in 4 cases and fair in 2 cases.At the last follow-up,the Harris score of hip joints was 85.1(range:75 to 93).Conclusions:Anterior medial fenestration in situ reduction and fixation surgery is feasible for the treatment of Pipkin Ⅰ and Ⅱ femoral head fractures. The short and midterm follow-up results reveal satisfactory effect.

Objectives:To explore the feasibility of anterior medial fenestration approach in situ reduction and fixation in the treatment of PipkinⅠ and Ⅱ femoral head fractures,and to explore the clinical effect of this operation.Methods:Hips of two anti-corrosion adult specimens treated with formalin were dissected, then anatomical structures and directional characteristics of anterior medial main muscles,ligaments,blood vessels and nerves were observed.The anterior medial fenestration approach was performed on bilateral hips of four fresh frozen specimens to determine pulling direction of stripped muscles and ligaments required during operation,and to observe and analyze vascular and nerve traction protection directions exposed in the approach.Determine extent of exposure to the approach and assess feasibility of this approach.The clinical data of 12 patients with Pipkin Ⅰ and Ⅱ femoral head fractures who underwent in situ reduction and fixation of anterior medial fenestration at Department of Orthopaedics,Affiliated Hospital of Chengdu University from February 2016 to April 2018 were retrospectively analyzed.There were 3 males and 9 females with an age of 48.5 years(range:37 to 59 years).There were 8 cases of Pipkin type Ⅰ and 4 cases of Pipkin type Ⅱ.The operation time,blood loss,fracture healing time,last Thompson-Epstein evaluation and Harris score were observed.Results:Anterior medial fenestration approach to expose the femoral head in 4 bilateral hips with a total of 8 sides of fresh frozen specimens.The upper boundary of observation fenestration was pubic body (anterior acetabulum),and the outer upper boundary was iliacus and psoas muscle.The lateral boundary was rectus femoris and femoral vessels,the lower boundary was transverse branch of the medial femoral circumflex artery and vein.The medial boundary was pubis muscle,short adductor muscle and long adductor muscle.Pubofemoral and iliofemoral ligament were seen in fenestration. Four quadrants in front of femoral head in fenestration can be seen after cutting switch capsule active hip joint. In 12 patients with femoral head fracture,the operation time was 107.5 minutes(range:90 to 135 minutes),and the intraoperative bleeding volume was 115.0 ml(range:85 to 150 ml).The patients were followed up for 18.6 months(range:12 to 28 months).The fracture healing time of 12 patients was 144.2 days(range:120 to 180 days).The curative effect was evaluated according to Thompson-Epstein standard at the last follow-up:excellent in 6 cases,good in 4 cases and fair in 2 cases.At the last follow-up,the Harris score of hip joints was 85.1(range:75 to 93).Conclusions:Anterior medial fenestration in situ reduction and fixation surgery is feasible for the treatment of Pipkin Ⅰ and Ⅱ femoral head fractures. The short and midterm follow-up results reveal satisfactory effect.

In response to the limitation of some medical image segmentation models for rectal cancer auxiliary diagnosis that are only applicable to single-modality images,a medical image segmentation method based on a cross attention mechanism that is applicable to both CT and MRI modalities is presented.Considering the different feature representations of CT and MRI images,a cross attention mechanism is proposed to unify the feature representations of the two types of images.In view of the small lesions on rectal cancer images,an improved Swin Transformer segmentation network with 3 branches is established,and the cross attention mechanism is incorporated into it,enabling the model to segment lesion areas in both types of images.The proposed method is validated using CT and MRI image data from patients with rectal cancer.Compared with ADDA,CycleGAN,and SIFA methods,the proposed method improves the accuracy by 2.94%,3.05%,0.71%on CT images,and 3.31%,4.55%,1.76%on MRI images,respectively,demonstrating its superior segmentation performance for both types of images.