Construction of the human hepatocellular carcinoma cell line stably expressed mutated hepatitis B virus X genes and changes of its biological behavior / 中国热带医学

@#Abstract: Objective　To construct HepG2, Huh7 cell lines stably express hepatitis B virus X (HBx) mutant (C1653T, T1753C), and explore their effect on the biological behavior of hepatocellular carcinoma cells. Methods　The lentivirus plasmid of pLVX-HBxC1653T-IRES-tdTomato, pLVX-HBxT1753C-IRES-tdTomato were obtained by PCR site mutagenesis according to wild type ayr HBx. Double enzyme digestion and Sanger sequencing were performed for accuracy of plasmid. Blank HepG2 and Huh7 cells were used as the control group, HepG2, Huh7 cells were infected by pLVX-HBx-IRES-tdTomato, pLVX-HBxC1653T-IRES-tdTomato, and pLVX-HBxT1753C-IRES-tdTomato lentivirus solution, then monoclonal cell was selected by 0.6 μg/mL puromycin. Immunostaining and Western Blot were performed for the verification of stable strains. CCK8 assay was performed for the proliferation capacity of stable strains. Western Blot was performed for expression of EMT-related signal molecules in cells. The independent samples t-test was used for comparison between two groups. Results Double enzyme digestion and Sanger sequencing showed that that the size of the cut fragments of recombinant lentiviral plasmids was correct, and the point mutation location and base substitution were correct, suggesting that the plasmid of pLVX-HBx-IRES-tdTomato, pLVX-HBxC1653T-IRES-tdTomato, pLVX-HBxT1753C-IRES-tdTomato were constructed successfully. Immunostaining and Western blot showed that HBX were expressed in stable strains, while there was no HBX expression in the blank control group, indicating that the HepG2 and Huh7 cell lines stably expressing HBx, HBxC1653T, HBxT1753C were successfully constructed. CCK8 assay showed that the proliferation capacity of HBx and mutant were enhanced compared to the control group (P<0.01), HBx C1653T displayed further additive the effect compared to HBx (P<0.05). Moreover, HBxC1653T mutation also significantly upregulated N-cadherin expression and downregulated E-cadherin expression, thus promoting the occurrence of EMT. Conclusions　HepG2 and Huh7 cell lines stably expressing HBx, HBxC1653T, HBxT1753C were successfully constructed, HBxC1653T mutation significantly enhanced the proliferation of HCC cells and epithelial to mesenchymal transition occurrence.

The Effect of FRAX on the Prediction of Osteoporotic Fractures in Urban Middle-aged and Elderly Healthy Chinese Adults

OBJECTIVE: We aimed to analyze the applicability of a fracture risk assessment tool for the prediction of osteoporotic fractures in middle-aged and elderly healthy Chinese adults. METHODS: A standard questionnaire was administered, and bone mineral density was measured in residents visiting the Dongliu Street Community Health Service Center. Paired t-tests were used to compare the FRAX-based probabilities of fractures estimated with and without consideration of bone mineral density. Risk stratification and partial correlation analyses were applied to analyze the associations between FRAX-based probabilities and body mass index or bone mineral density at different sites. RESULTS: A total of 444 subjects were included in this study. Of these subjects, 175 (39.59%) were diagnosed as osteoporotic, and 208 (47.06%) were diagnosed as osteopenic. The Kappa value for the detection of osteoporosis at the L1-L4 lumbar spine and femoral neck was 0.314. The FRAX-based 10-year major osteoporotic fracture probability and hip osteoporotic fracture probability estimated without considering bone mineral density were 4.93% and 1.64%, respectively; when estimated while considering bone mineral density, these probabilities were 4.97% and 1.54%, respectively. A significant positive association was observed between the FRAX-based fracture probabilities estimated with and without consideration of bone mineral density, while significant negative associations between body mass index and the estimated FRAX-based fracture probabilities after adjustment for age and the estimated FRAX-based fracture probabilities and femoral neck bone mineral density were identified. These results remained the same after controlling for lumbar spine bone mineral density. CONCLUSIONS: The Chinese FRAX model could predict osteoporotic fracture risk regardless of whether bone mineral density was considered and was especially appropriate for predicting osteoporotic fractures of the femoral neck.

An unusual presentation of thoracic para-aortic hematoma due to blunt trauma.

An 11-yr-old boy was admitted to the hospital with gradually aggravating acute abdominal pain along with nausea, vomiting and constipation for 5 days. The pain started after blunt trauma on the superior abdomen. He had normal laboratory tests and abdominal examination, but his chest radiograph showed infiltration in the right lung and an abnormal mass on the left diaphragm. Further investigation like computed tomography (CT) and magnetic resonance MR angiography, revealed an abnormal para-aotic mass located a mass located para-aortic above posterior to the left diaphragm with a well-defined margin. The abnormal mass was finally confirmed to be a hematoma with exploratory thoracotomy. The rarity of this kind of presentation is discussed for making an early and correct diagnosis.