Abstract: Objective　To understand the contamination status, drug resistance, virulence gene carrying status, and molecular typing characteristics of Vibrio parahaemolyticus (VP) in aquatic products sold in Nanchang City. Methods　A total of 170 commercial crayfishes, freshwater fish frogs and related smears samples were collected from various farmers' markets in Nanchang from March to September 2021. The strains of V. parahaemolyticus were detected and isolated from the samples. Antibiotic resistance test, virulence gene test, and pulsed-field gel electrophoresis (PFGE) molecular typing analysis were carried out. Results　Among the collected samples, V.parahaemolyticus was only isolated from crayfish and crayfish smear samples, with a total of 35 strains of VP isolated. No V.parahaemolyticus strain was isolated from other freshwater fish, frogs, and their smear samples. Among the 17 common antibiotics tested, only two trains showed resistance to ampicillin, and one strain to streptomycin, , and all were sensitive to other antibiotics; all 35 strains of V. parahaemolyticus carried the gene, but only one strain carried the heat-resistant related hemolysin gene trh, and no direct heat-resistant hemolysin gene tdh positive strain was found; PFGE pattern clustering showed that there was no strain with the same PFGE pattern, and there was no obvious dominant cluster among these strains, and their genetic relationship was relatively distant. Conclusions　The contamination of V. parahaemolyticus in small and medium-sized crayfish sold in the market in Nanchang City is relatively serious. The V. parahaemolyticus isolates in these polluted crayfish generally do not carry key virulence genes such as tdh, are sensitive to common antibiotics, and only have low-level resistance to ampicillin and streptomycin. PFGE pattern clustering showed that V. parahaemolyticus does not have no obvious dominant cluster, and these strains have rich genetic diversity, indicating that they may have different sources.

Objective To investigate the biological characteristics of monoclonal antibodies against human liver cancer stem cells and its therapeutic effect in combination with cisplatin in the treatment of hepatocellular carcinoma. Methods Cell culture in serum?free medium and PKH26 staining were used to determine the existence of cancer stem cells in human liver Bel7402?V3 cell line. The co?expression of antigen recognized by monoclonal antibody ( McAb ) 15D2 and epithelial specific antigen ( ESA ) and PKH26?positive cells in the Bel7402?V3 cells were detected by immunofluorescence assay. Serum?free suspension culture was used to detect the self?renewal ability of 15D2?positive Bel7402?V3 cells sorted by flow cytometry and the effect of 15D2 on the self?renewal ability of Bel7402?V3 cells. The effect of 15D2 on cisplatin resistance in the cells was examined by CCK8 method. The inhibitory effect of 15D2 combined with cisplatin on the transplanted tumor growth in mice was also observed. Results Single PKH26?positive cells were observed in the Bel7402?V3 cell spheroids cultured for 11 days. Immunofluorescence assay showed that the 15D2?recognized antigen could be conjugated with PKH26 and ESA and co?localized on Bel7402?V3 cells. The spheroid formation rate of 15D2?positive cells in serum?free medium was significantly higher than that of 15D2?negative cells [(30.4±3.4)% vs. (8.8±1.8)%,P<0.01]. The cisplatin resistance of 15D2?positive cells was obviously higher than that of 15D2?negative cells (IC50:1.014μmol/L vs. 0.365μmol/L). McAb 15D2 significantly suppressed the spheroid formation of Bel7402?V3 cells, with an inhibition rate of 37.5%. McAb 15D2 also notably inhibited the cisplatin resistance of Bel7302?V3 cells. The IC50 was 0.211μg/ml in the 15D2 group and 0. 325 μg/ml in the control group. The mouse experiment showed that the tumor growth rates of 50 mg/kg, 25 mg/kg and 12.5 mg/kg 15D2?treatment groups were 82.6%, 71.4% and 60.0%, respectively; that of the 50 mg/kg 15D2 + cisplatin group was 91. 0%, and that of the cisplatin monotherapy was 56. 7%. Conclusion McAb 15D2 is a functional monoclonal antibody targeting liver cancer stem cells, which could be a potential monoclonal antibody drug for the stem cell?targeted therapy of liver cancer.

Objective To investigate the biological characteristics of monoclonal antibodies against human liver cancer stem cells and its therapeutic effect in combination with cisplatin in the treatment of hepatocellular carcinoma. Methods Cell culture in serum?free medium and PKH26 staining were used to determine the existence of cancer stem cells in human liver Bel7402?V3 cell line. The co?expression of antigen recognized by monoclonal antibody ( McAb ) 15D2 and epithelial specific antigen ( ESA ) and PKH26?positive cells in the Bel7402?V3 cells were detected by immunofluorescence assay. Serum?free suspension culture was used to detect the self?renewal ability of 15D2?positive Bel7402?V3 cells sorted by flow cytometry and the effect of 15D2 on the self?renewal ability of Bel7402?V3 cells. The effect of 15D2 on cisplatin resistance in the cells was examined by CCK8 method. The inhibitory effect of 15D2 combined with cisplatin on the transplanted tumor growth in mice was also observed. Results Single PKH26?positive cells were observed in the Bel7402?V3 cell spheroids cultured for 11 days. Immunofluorescence assay showed that the 15D2?recognized antigen could be conjugated with PKH26 and ESA and co?localized on Bel7402?V3 cells. The spheroid formation rate of 15D2?positive cells in serum?free medium was significantly higher than that of 15D2?negative cells [(30.4±3.4)% vs. (8.8±1.8)%,P<0.01]. The cisplatin resistance of 15D2?positive cells was obviously higher than that of 15D2?negative cells (IC50:1.014μmol/L vs. 0.365μmol/L). McAb 15D2 significantly suppressed the spheroid formation of Bel7402?V3 cells, with an inhibition rate of 37.5%. McAb 15D2 also notably inhibited the cisplatin resistance of Bel7302?V3 cells. The IC50 was 0.211μg/ml in the 15D2 group and 0. 325 μg/ml in the control group. The mouse experiment showed that the tumor growth rates of 50 mg/kg, 25 mg/kg and 12.5 mg/kg 15D2?treatment groups were 82.6%, 71.4% and 60.0%, respectively; that of the 50 mg/kg 15D2 + cisplatin group was 91. 0%, and that of the cisplatin monotherapy was 56. 7%. Conclusion McAb 15D2 is a functional monoclonal antibody targeting liver cancer stem cells, which could be a potential monoclonal antibody drug for the stem cell?targeted therapy of liver cancer.