AIM: To study the effect of hypoxia on CD73 expression in mouse microvascular endothelial cell line bEnd.3. METHODS: ① bEnd.3 cells were exposed to different periods of hypoxia. ② Concentration of LDH released by bEnd.3 cells into the culture medium was detected. ③ Surface CD73 activity in bEnd.3 cells was measured by HPLC according to the conversion of E-AMP to E-ADO. ④ CD73 mRNA expression were analyzed by semiquantitative RT-PCR. ⑤ Cell surface proteins were biotinylated and CD73 was detected by avidin blots of immunoprecipitation with mAb TY23. RESULTS: ① bEnd.3 cells exposed to hypoxia for 24 h demonstrated a significant increase in LDH release (P

AIM: To study the role of protein kinase B (PKB) in tryptase-induced gene expression on ECV304 cells. METHODS: The expression of PKB, transcript factor AP-1 and NF-?B P65, IL-8, JNK, p38MAPK, and the activity of PKB were measured using RT-PCR and Western blotting. RESULTS: Tryptase at concentration of 1 ?g/L increased the activity of PKB by promoting PKB phosphorylation, promoted the expression of PKB, chemokine IL-8, transcription factor AP-1 and NF-?B P65, however, no changes of JNK and p38MAPK was observed. PI3K specific inhibitor (LY294002) abolished the augment of PKB, NF-?B P65 and IL-8 expression. Antisense PKB cDNA transfection also abolished the augment of PKB, AP-1, NF-?B P65 and IL-8 expression. Though PAR2 antibody did not inhibit PKB expression, it did inhibit the phosphorylation by tryptase in ECV304 cells. CONCLUSION: These results indicate that tryptase can activate PKB through PAR2 receptor and subsequently NF-?B, AP1, IL-8 and PKB expression.

Objective To detect steroidogenic acute regulatory protein (StAR) expression in apolipoprotein E-deficient mice at different ages and serum lipid levels. Methods Nighty-six C57BL/6J and apoE-/- mice were enrolled, which were divided into 16 groups with 6 mice per group according to age (1 day, 1, 3, 5 months), sex and genotype (C57BL/6J and apoE-/-). The serum lipid levels in C57BL/6J and apoE-/- mice were detected by commercial kits. StAR mRNA and protein expressions in liver were detected by Real-time PCR and Western blot respectively. Results ApoE-/- mice had higher LDL-cholesterol and lower HDL-cholesterol compared with C57BL/6J mice of the same age and sex. StAR mRNA and protein expressions were decreased following with aging in C57BL/6J mice. However, in apoE-/- mice with higher lipid levels, StAR mRNA and protein expressions were changed with the lipid levels other than ages. StAR mRNA and protein increased in the early stage, and then decreased with the increasement of lipids levels. Conclusions StAR could affect lipids levels and may be an effective regulator for atherosclerosis and other cardiovascular diseases.

OBJECTIVETo investigate the antitumor and anti-metastatic effect of in situ transduction of adenovirus encoding cytosine deaminase (AdCD) followed by the systemic use of 5-fluorocytosine (5-FC) in the orthotopic (o.t.) prostate cancer mouse model.

METHODSThe o.t. prostate cancer model of C57BL/6 mouse was developed by o.t. inoculation of RM-1 cells to the subcapsular area of the prostate gland. In situ transduction of the CD gene, followed by systemic use of 5-FC at a daily dosage of 300 mg/kg for 14 days, was performed two days later.

RESULTSCompared with mice treated with Adbeta-gal/5-FC, 5-FC and PBS, mice of the o.t. model receiving in situ treatment of AdCD/5-FC had significant prolongation of survival and suppression of local tumor growth. More importantly, pathological observations showed that metastatic activity occurred in all mice of the PBS, 5-FC and Adbeta-gal groups including metastasis to the iliac lymph node (10/10, 10/10, 10/10) and the lung (8/10, 7/10, 7/10). However, only two out of ten had iliac lymphatic metastasis in the AdCD/5-FC group with no systemic or preaotic lymphatic metastasis, suggesting a strong metastatic inhibitory effect.

CONCLUSIONSIn situ transduction of AdCD followed by systemic use of 5-FC leads to the inhibitory effect on tumor growth and metastatic activity in the o.t. mouse model of prostate cancer. Clinically, it may be possible to treat metastatic or recurrent prostate cancer with a novel gene therapy using in situ injection techniques in future.

Adenoviridae ; genetics ; Animals ; Cell Division ; drug effects ; genetics ; Cytosine Deaminase ; Disease Models, Animal ; Flucytosine ; pharmacology ; Lymphatic Metastasis ; genetics ; pathology ; prevention & control ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Nucleoside Deaminases ; genetics ; Prostatic Neoplasms ; genetics ; mortality ; prevention & control ; Survival Rate ; Transfection ; Tumor Cells, Cultured

AIM:To study the effect of CD73 on breast cancer cell line MB-MDA-231 adhesion to extracellular matrix.METHODS: ① CD73 siRNA plasmid was constructed and transfected into MB-MDA-231 cells by lipofectamine 2000.② The transfection efficiency was analyzed by flow cytometry using eGFP as a marker gene.Stable transfected MB-MDA-231 cells were selected using G418.③ RT-PCR and Western blotting analysis of CD73 expression in MB-MDA-231 cells was performed.④ The effects of CD73 on MB-MDA-231 cells adhesion to extracellular matrix were assessed by cell adhesion assay.RESULTS: ① Transfection of MB-MDA-231 cells with CD73 siRNA plasmid significantly inhibited CD73 expression at mRNA level.The efficiency was up to 91%.② Transfection of MB-MDA-231 cells with CD73 siRNA plasmid significantly inhibited CD73 expression at a protein level.The efficiency was up to 79.3%.③ Treatment of MB-MDA-231 cells with CD73 siRNA resulted in diminished adhesion to extracellular matrix.CONCLUSION: This study demonstrates that CD73 siRNA effectively inhibits CD73 gene expression in MB-MDA-231 cells leading to adhesion to extracellular matrix suppression.

After more than3 years of pilot (since 2010), standardized training of resident physicians in Shanghai has made great progress, however, it still needs to be improved. Cerebral and spinal vascular diseases are main types of neurological diseases and they seriously influence people's life and health. Interventional neuroradiology plays an important role in the diagnosis and treatment of cerebral and spinal vascular diseases. However, in the current residency training system in Shanghai or other provinces of China, the training of interventional neuroradiology is not included. In this paper, the authors analyzed the important role of interventional neuroradiology in diagnosis and treatment of cerebral and spinal vascular disorders and discussed the feasibility, method, and time arrangement of including interventional neuroradiology curriculum into the schedule of neurology standardizedresidency training and hoped to provide reference for the relevant departments to formulate a future policy.

Objective To construct lentivirus vectors carrying 16 short hairpin RNA (shRNA) expression cassettes targeting histone acetyltransferases and provide a powerful research approach to explore the mechanism of epigenetic genes in regulating hepatitis B virus (HBV).Methods Following the rule of short shRNA primer design,eight-pair primers (A ~ H )for each gene,which had stable interfering efficiency,were designed.The annealed primers were connected to the empty lentiviral vectors of shRNA for transformation.In order to confirm the positive clones,clones were analyzed by real-time polymerase chain reaction (RT-PCR ).Then, qualified plasmids were verified by enzyme digestion technology.Four shRNA lentivirus plasmids against the same gene were mixed to build lentivirus respectively.After the virus transfected into 293T cells for 48 and 72 hours,supernatants were collected to infect HepG2.2.15 cells.The percentage of fluorescent cells were observed and assessed by microscope 72 hours after infection.Results One hundred and twenty-eight lentiviral vectors of RNA interference (RNAi)library were constructed against 16 histone acetyltransferases and more than 80% of HepG2.2.15 cells were infected with lentivirus 72 hours after infection.Conclusions Sixteen shRNA lentivirus vectors against histone acetyltransferase are successfully constructed.Thus,a solid foundation for the study of the effect of human histone deacetylase on HBV replication is established.

To analyze and evaluate the knowledge of Chinese Guidelines of Diabetes Prevention and Treatment in Shanghai medical staff. 175 medical staff working in endocrinology or community health were enrolled and evaluated by a questionnaire of guidelines about the state of professional, training, and related knowledge. Only 16. 6% medical staffwere trained about the guidelines( 46. 67% from the general hospitals, 14. 75% from secod-level hospital and 7. 14% persons from the community hospitals, P＜0. 01 ). The total correct answer rate of the guidelines was 37. 36%. The correct rate of community hospitals was lower than others( P＜0. 05 ). The rate of doctors' was higher than nurses'( P＜0. 05 ). There were difference between doctors and nurses with the key point of diabetes care knowledge in different level hospitals. The effective method of clinical training in diabetes care should be explored. We still have to work hard to promote the effect of diabetes control and prevention. Effective training about the guidelines should be enhanced. The cooperation between general hospitals and community health institutions in diabetes prevention and treatment should be enhanced.

Objective:To investigate the efficacy and safety of ixazomib combined with lenalidomide and dexamethasone (IRd) regimen in treatment of multiple myeloma (MM) patients in the real world practice.Methods:The clinical data of 24 MM patients treated with IRd regimen from January 2019 to January 2021 in the Union Hospital, Tongji Medical College, Huazhong University of Science and Technology were retrospectively analyzed, and their efficacy and adverse reactions were analyzed. Among the 24 patients, 5 patients were relapsed and refractory (relapsed/refractory group), and 19 newly treated patients (conversion group) who responded to bortezomib induction therapy but converted to IRd regimen due to adverse reactions or other reasons.Results:The 24 patients were treated for a median of 4 cycles (2-7 cycles), with 8 cases of complete remission (CR), 6 cases of very good partial remission (VGPR), 8 cases of partial remission (PR), 1 case of disease progression (PD), 1 case of minimal response (MR), and the overall response rate (ORR) was 91.7% (22/24); the median progression-free survival (PFS) time was 15 months (95% CI 6.6-23.4 months); 6 CR patients were negative for minimal residual disease (MRD). The common adverse reactions were hematological adverse reactions, peripheral neuropathy, fatigue, gastrointestinal reactions, and infections. The incidence rate of grade 3-4 adverse reactions was 25.0% (6/24). In the relapsed/refractory group, the best efficacy was VGPR in 1 case, PR in 3 cases, and MR in 1 case, all patients withdrew from the IRd regimen therapy due to PD after transient remission or poor effect; in the conversion group, the best efficacy was CR in 8 cases, VGPR in 5 cases, PR in 5 cases, and PD in 1 case, 57.9% (11/19) patients maintained their original best response, and 36.8% (7/19) patients improved their best response to CR; the difference in median PFS time between the two groups was statistically significant (7 months vs. not reached, P = 0.018). Conclusions:The IRd regimen is safe and effective for MM patients, especially for the conversion patients after effective bortezomib induction therapy. Although patients with relapsed/refractory MM who have previously used multi-line therapy respond to IRd regimen, the duration of remission is limited.