Objective To transfect bone marrow mesenchymal stem cells (BMSCs) of rats by recombinant adenovirus Ad-human matrix metalloproteinase 1 (hMMP-1) in vitro so as to lay the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation. Methods BMSCs were isolated from bone marrow of 2-3 weeks old Sprague Dawley rats by whole bone marrow adherence method and identified, then transfected by recombinant adenovirus Ad-hMMP-1 carrying enhanced green fluorescent protein (EGFP) marker in vitro. The green fluorescent expression was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry to determine the optimum multiplicity of infection (MOI). BMSCs at passage 3 were divided into 3 groups: untransfected BMSCs group (group A), Ad-EGFP transfected BMSCs group (group B), and Ad-hMMP-1-EGFP transfected BMSCs group (group C); the gene and intracellular protein of hMMP-1 were detected by RT-PCR and Western blot; the ELISA assay was used to detect the supernatant protein expression, and the hMMP-1 activity was measured by fluorescent quantification kit. Results The green fluorescent was observed in BMSCs transfected by recombinant adenovirus at 24 hours after transfection; the fluorescence intensity was highest at 72 hours; and the optimum MOI was 200. The cells of 3 groups entered the logarithmic growth phase on the 3rd day and reached plateau phase on the 6th day by MTT assay; no significant difference was found in the cell proliferation rate among 3 groups (P gt; 0.05). RT-PCR, Western blot, and ELISA assay showed high expressions of the hMMP-1 gene and protein in group C, but no expression in groups A and B. The hMMP-1 activity was 1.24 nmol/(mg · min) in group C, but hMMP-1 activity was not detectable in groups A and B. Conclusion The exogenous hMMP-1 gene is successfully transfected into BMSCs of rats via recombinant adenovirus and can highly express, which lays the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP-1 gene transplantation.
ObjectiveTo evaluate the clinical value of color Doppler ultrasound in diagnosing prehepatic portal hypertension. MethodsA retrospective analysis was performed to analyze the results of color Doppler ultrasonography in 9 patients with prehepatic portal hypertension diagnosed between June 2012 and January 2015, including vessel diameter, shape, nature and direction of blood flow, and fistula blood flow spectrum. ResultsAmong the 9 patients, the color Doppler ultrasound found 3 patients with regional portal widened, increased and faster blood flow with the emergence of low-impedance spectrum artery, splenic vein widened with returning blocked and flocculent substance within the splenic vein lumen, irregular or streak-shaped low weak echo during splenic vein reduction, and unstable or weakened blood flow velocity. Two patients were confirmed with splenic vein thrombosis by ultrasound and other imaging methods with significantly reduced blood in splenic vein. For the other four patients with regional portal hypertension, obvious abnormalities in portal system were not detected by color Doppler ultrasound, but they were checked with other methods. The ultrasound positive diagnosis of the 9 patients was 5, with 4 missed. ConclusionThe color Doppler ultrasound has some values in screening, diagnosis and follow-up of prehepatic portal hypertension, but it can also be influenced by many factors with a high missed diagnosis rate. Carefully observing the portal system lumen structure, internal echo and blood flow combined with other imaging studies, and emphasizing clinical history of the patients can further improve diagnostic accuracy.