Mice have been widely used in the study of primary liver cancer owing to the close similarity of its genome to that of humans,its strong reproductive ability,the low cost of model construction,and the ease of genetic manipulation,including molecular mechanisms of pathogenesis and potential drug targets.Traditional animal models are increasingly falling short of meeting the needs of precision medicine research because of their inability to reproduce tumor microenvironment interactions and control the specificity of molecular subtypes.This study systematically compared the technical advantages of tail vein high-pressure injection,combined with transposon system(HTVI-TS),with traditional models in liver cancer research,and focused on the application value of the HTVI-TS model in the mechanism study of tumorigenesis and development,immunotherapy response prediction,and individualized evaluation of targeted drugs.This report presents a new research platform for precise diagnosis and treatment of primary liver cancer by simulating the heterogeneous evolution process of the cancer.The findings provide a theoretical basis for optimizing the selection of preclinical research models for liver cancer;the expansion potential of this technology in liver cancer research is outlined.

Mice have been widely used in the study of primary liver cancer owing to the close similarity of its genome to that of humans,its strong reproductive ability,the low cost of model construction,and the ease of genetic manipulation,including molecular mechanisms of pathogenesis and potential drug targets.Traditional animal models are increasingly falling short of meeting the needs of precision medicine research because of their inability to reproduce tumor microenvironment interactions and control the specificity of molecular subtypes.This study systematically compared the technical advantages of tail vein high-pressure injection,combined with transposon system(HTVI-TS),with traditional models in liver cancer research,and focused on the application value of the HTVI-TS model in the mechanism study of tumorigenesis and development,immunotherapy response prediction,and individualized evaluation of targeted drugs.This report presents a new research platform for precise diagnosis and treatment of primary liver cancer by simulating the heterogeneous evolution process of the cancer.The findings provide a theoretical basis for optimizing the selection of preclinical research models for liver cancer;the expansion potential of this technology in liver cancer research is outlined.

Objective To analyze the confirmation results of initial screening positive for human immunodeficiency virus (HIV) antibodies and to investigate the rationality of the gray zone for the signal-to-cutoff (S/CO) ratio values obtained by chemiluminescence assay. Methods A total of 184 specimens with positive HIV antibody screening results were collected. Confirmation was conducted using the Western blot (WB) method and viral nucleic acid testing, and the confirmation outcomes and band distribution were observed. The receiver operating characteristic (ROC) curve was employed to predict the optimal cutoff value, and the rationality of S/CO values in establishing a gray zone for HIV antibody was analyzed. Results Among the 184 HIV antibody screening positive samples, 153(83.15%) were initially screened by chemiluminescence, and 31 (16.85%) were screened by colloidal gold method. Male cases (57.07%) and those aged 40 years or older (75.00%) had higher proportion of initial screening positivity than females and other age groups. Supplemental WB testing revealed that 101 (54.89%) cases were confirmed as HIV-1 antibody positive, 56(30.43%) were negative, and 27(14.67%) were indeterminate. Of the 27 indeterminate cases, one was confirmed positive by NAT (initial S/CO value by chemiluminescence5.60), while the remaining 26 were negative. The positive agreement rates between chemiluminescence, colloidal gold and supplemental WB testing were 65.36% and 3.23%, respectively, with statistically significant difference (χ²=40.191, P < 0.05). Among 27 cases with indeterminate HIV-1 antibody results, upon further nucleic acid testing for viral load, one case exhibited WB banding pattern of P24+gp160, and the nucleic acid load confirmed this case as positive. In the distribution of band patterns among indeterminate samples, P24 had the highest occurrence (74.07%), followed by P66 (11.11%), gp160 (7.41%) as well as P17 and gp41 (both 3.70%). The ROC curve predicted the optimal S/CO cutoff value to be 5.15, with an area under the curve (AUC) of 0.904, sensitivity and specificity of 83.30% and 76.40%, respectively, and 95%CI of 0.861 to 0.948. There was a good correlation between the WB band patterns and confirmation results for initial screening positive cases (P < 0.05), with the highest correlation found for the P24 band (r=0.910). Conclusion There is a certain rate of false positives among HIV antibody initial screening positive cases, and effective screening can be achieved through WB testing. Additionally, based on the current testing platform, the gray zone for HIV antibody S/CO values in the chemiluminescence assay is set at 1.00 to 5.15, which exhibits high sensitivity.

Objective:To further improve the understanding of paroxysmal nocturnal hemoglobinuria (PNH), we retrospectively analyzed and summarized the clinical characteristics, treatment status, and survival status of patients with PNH in Zhejiang Province.Methods:This study included 289 patients with PNH who visited 20 hospitals in Zhejiang Province. Their clinical characteristics, comorbidity, laboratory test results, and medications were analyzed and summarized.Results:Among the 289 patients with PNH, 148 males and 141 females, with a median onset age of 45 (16-87) years and a peak onset age of 20-49 years (57.8% ). The median lactic dehydrogenase (LDH) level was 1 142 (604-1 925) U/L. Classified by type, 70.9% (166/234) were classical, 24.4% (57/234) were PNH/bone marrow failure (BMF), and 4.7% (11/234) were subclinical. The main clinical manifestations included fatigue or weakness (80.8%, 235/289), dizziness (73.4%, 212/289), darkened urine color (66.2%, 179/272), and jaundice (46.2%, 126/270). Common comorbidities were hemoglobinuria (58.7% ), renal dysfunction (17.6% ), and thrombosis (15.0% ). Moreover, 82.3% of the patients received glucocorticoid therapy, 70.9% required blood transfusion, 30.7% used immunosuppressive agents, 13.8% received anticoagulant therapy, and 6.3% received allogeneic hematopoietic stem cell transplantation. The 10-year overall survival (OS) rate was 84.4% (95% CI 78.0% -91.3% ) . Conclusion:Patients with PNH are more common in young and middle-aged people, with a similar incidence rate between men and women. Common clinical manifestations include fatigue, hemoglobinuria, jaundice, renal dysfunction, and recurrent thrombosis. The 10-year OS of this group is similar to reports from other centers in China.

Objective:To evaluate the efficacy and safety of hypomethylating agents (HMA) in patients with myelodysplastic syndromes (MDS) .Methods:A total of 409 MDS patients from 45 hospitals in Zhejiang province who received at least four consecutive cycles of HMA monotherapy as initial therapy were enrolled to evaluate the efficacy and safety of HMA. Mann-Whitney U or Chi-square tests were used to compare the differences in the clinical data. Logistic regression and Cox regression were used to analyze the factors affecting efficacy and survival. Kaplan-Meier was used for survival analysis. Results:Patients received HMA treatment for a median of 6 cycles (range, 4-25 cycles) . The complete remission (CR) rate was 33.98% and the overall response rate (ORR) was 77.02%. Multivariate analysis revealed that complex karyotype ( P=0.02, OR=0.39, 95% CI 0.18-0.84) was an independent favorable factor for CR rate. TP53 mutation ( P=0.02, OR=0.22, 95% CI 0.06-0.77) was a predictive factor for a higher ORR. The median OS for the HMA-treated patients was 25.67 (95% CI 21.14-30.19) months. HMA response ( P=0.036, HR=0.47, 95% CI 0.23-0.95) was an independent favorable prognostic factor, whereas complex karyotype ( P=0.024, HR=2.14, 95% CI 1.10-4.15) , leukemia transformation ( P<0.001, HR=2.839, 95% CI 1.64-4.92) , and TP53 mutation ( P=0.012, HR=2.19, 95% CI 1.19-4.07) were independent adverse prognostic factors. There was no significant difference in efficacy and survival between the reduced and standard doses of HMA. The CR rate and ORR of MDS patients treated with decitabine and azacitidine were not significantly different. The median OS of patients treated with decitabine was longer compared with that of patients treated with azacitidine (29.53 months vs 20.17 months, P=0.007) . The incidence of bone marrow suppression and pneumonia in the decitabine group was higher compared with that in the azacitidine group. Conclusion:Continuous and regular use of appropriate doses of hypomethylating agents may benefit MDS patients to the greatest extent if it is tolerated.

Objective:Cushing′s disease(CD) is caused by the pituitary adrenocorticotroph hormone(ACTH) secreting adenomas, leading to increased serum cortisol levels and various abnormal metabolic processes. Untreated CD is linked to high mortality, thus it is critical to elucidate its pathogenesis. This study aims to explore the pathogenesis of pituitary ACTH adenomas using whole-genome sequencing analysis.Methods:Fresh tumor tissues and peripheral blood samples were collected in 9 confirmed cases of pituitary ACTH adenomas who underwent surgery. Whole genome sequencing was then performed, followed by analysis and verification of single nucleotide mutations, copy number variation(CNV) and chromosome structure variations.Results:Somatic USP8 mutations(p.Ser718del, p. Ser718Pro, p. Pro720Arg, p. Pro720Gln) were found in 5 patients, with a rate of 55.6%; CNV of USP8 was detected in 1 patient; TP53(p.Cys135Tyr), NF1(p.Val1049Glufs*11) and KMT2C(c.3323+ 1G>A) mutations were identified in 1 patient harboring wild-type USP8. CNV analysis showed a loss of heterozygosity in multiple chromosomes in a wild-type USP8 patient. Structural variations were found in 2 with unknown significance. No germline gene mutations were detected in this study.Conclusion:Somatic USP8 mutations, increased copy number of USP8, variations of tumor-related genes such as TP53 and extensive somatic CNV all contribute to pathogenesis of CD. Chromosomal structure variations may suggest high-risk pituitary ACTH adenomas, and call for frequent follow-up and aggressive treatment.

Breast cancer is one of the most common malignant tumors in females.Recent years,surgery,chemotherapy as well as other systemic therapy had greatly improved the prognosis of the patients.However,damage of ovarian function by chemotherapy lowered life quality,especially for young females.At present,there are several methods to protect the ovarian function of female patients undergoing chemotherapy,such as administration of a gonadotropin-releasing hormone (GnRH) analogs,ovarian cryopreservation,unfertilized ova cryopreservation,embryo cryopreservation,inhibitors of apoptosis,etc.Each method has its advantage,disadvantage and indications.Issues related to ovarian protection are reviewed here.

Objective:To detect and verifica the gene profile difference of microRNA-146a (miR-146a) and its role in the pro-liferation of vascular smooth muscle cells (VSMCs) by gene chip technology. Methods: Artificially synthesized miR-146a mimics(50 nmol/L) ,miR-146 inhibitor ( 50 nmol/L ) , scramble ( 50 nmol/L ) and PBS were transfected into cultured primary rat VSMCs in vitro. After transfection,Real time PCR was used to measure the levels of miR-146a and the cell counting kit 8(CCK8) was employed to investigate the proliferation of VSMCs. The VSMCs interfered by miR-146a inhibitor or miR-146a control were examined by gene chips and the profile of gene were analyzed by bioinformatics technology to detect the different genes and signal transduction pathway. The changes in mRNAs and proteins were accessed separately by Real time PCR and Western blot. Results: Compared with sham and control VSMCs,miR-146a expression level was significantly decreased in treatment with miR-146a inhibitor(P<0. 01),as well as optical density(OD) was also shown remarkably down regulated simultaneously(P<0. 05). The investigation of gene profile revealed that the p53 signal pathway was up-regulated in VSMCs interfered by miR-146a. The mRNA and protein expression levels of p53, caspase3 and PTEN in p53 signal transduction pathway didn′t show significant differences(P>0. 05),however,the mRNA and protein expression levels of cyclin D1 significantly increased in treatment with miR-146a mimics VSMCs group and decreased in miR-146a inhibitor VSMCs group ( compared with sham VSMCs group, both P<0. 05 ) . Conclusion: Our data indicated that miR-146a may promote the proliferation of rat VSMCs by up-regulating cyclin D1 expression.

Objective:To investigate the feasibility of real-time fluorescence quantitative PCR in the identification of Fritillariae Cirrhosae Bulbus.Methods:The DNA of samples was extracted by magnetic beads ,the primers were amplified by real-time fluores-cence quantitative PCR , and the Cq value and amplification curve were used to determine the samples .Results:The Cq values for five batches of Fritillariae Cirrhosae Bulbus were lower than 30, and the curve had obvious growth period .No Cq value was shown for Fritil-lariae Ussuriensis Bulbus , Fritillariae Thunbergii Bulbus and Bolbostemmatis Rhizoma with straight line curves .Conclusion:The meth-od is simple,feasible and effective in the identification of Bulbus Fritillariae Cirrhosae with high accuracy and good reproducibility .

AIM:To investigate the proliferation property of stable chemerin gene knockdown vascular smooth muscle cells ( VSMCs) and to explore its mechanism .METHODS:The normal VSMCs , chemerin gene interfering control VSMCs and stable chemerin gene knockdown VSMCs were divided into normal group , PDGF group, control group and knockdown group .The VSMCs in PDGF group were given platelet-derived growth factor-BB ( PDGF-BB) to initiate proli-feration.The cell counting and BrdU assay were employed to investigate the proliferation property of VSMCs .The mitogen-activated protein kinase ( MAPK) signal pathway was determined by Western blot .RESULTS:The cell number and BrdU A value in PDGF-BB-treated VSMCs significantly increased as compared with the normal VSMCs ( P<0.05 ) .Compared with the normal VSMCs , the chemerin knockdown VSMCs showed obviously decreased cell number and BrdU A value ( P<0.05).Simultaneously, no significant difference in the proliferation of VSMCs between the normal VSMCs and the control VSMCs was observed.No significant difference of the protein levels of p-ERK1/2, ERK1/2, p-p38 MAPK and p38 MAPK among 4 kinds of VSMCs was found .The protein level of p-JNK in PDGF-BB-treated VSMCs was up-regulated, while it was down-regulated in chemerin knockdown VSMCs compared with the normal VSMCs .CONCLUSION: Chemerin pro-motes the proliferation of mouse vascular smooth muscle cells by up-regulating p-JNK production .