Résumé
To establish method suitable to assay HCMVpp65 of the blood donors in blood bank and to supply safe blood to the patients, the immunocytochemical techniques were used, (6 - 8) x 10(6)/ml cells were counted, 50 x 10(3) cells were detected by light microscope, The results showed that 10 positive samples in 103 samples were found, positive rate was 9.71%, among 10 positive samples, 2 samples were still positive in the second detecting. In conclusion, this method is simple, quick and effective, suitable to detect HCMVpp65 of the blood donors in the blood bank.
Sujets)
Femelle , Humains , Mâle , Banques de sang , Donneurs de sang , Immunohistochimie , Phosphoprotéines , Sang , Protéines de la matrice virale , SangRésumé
Objective To evaluate the effect of pancreatic elastase on the expression of TNF ? and IL 1? in Kupffer cells induced by lipopolysacchride. Method Cultivating Kupffer cells were divided into 3 groups. In group A, physiological saline was added into the culture medium as control (control group). Lipopolysacchride (LPS) was added instead of saline in group B (LPS group). In group C both lipopolysacchride and pancreatic elastase were added to the cultere medium (LPS+elastase group). The expressions of TNF ?, IL 1? and TLR4 mRNA in Kupffer cells were determined by RT PCR, and concentrations of TNF ? and IL 1? in the culture media by ELISA. Results The results of both RT PCR and ELISA indicated that the expressions of TNF ? and IL 1? in group C was significantly higher than that in group B. Conclusion It was concluded that pancreatic proteases such as elastase could enhance the expressions of TNF ? and IL 1? of hepatic Kupffer cells induced by lipopolysacchride, and the results the might offer the explanation why inflammatory reaction could be amplified in the course of acute pancreatitis
Résumé
AIM: Gene transduction with a recombined murine stem cell retroviral vector has been investigated to find an effective way of gene transduction and to offer theory and experimental basis for the recombined murine stem cell retroviral vector used for gene transduction. METHODS: 1. Construction of retrovirus vector: EC1-4 gene (repeats 1-4 of cadherin-5 extracellular domain) and mutant (Ser 222A) MEK1 gene were cloned into retrovirus vector pMSCV after cut by Bgl Ⅱ and EcoR 1 restriction endonuclease. 2. Obtaining CD41 + cells and cell culture: Cells expressing CD34 + from cord blood were isolated. The inducement of cells expressing CD41 from CD34 + cells was performed by using TPO and cells CD41 + were selected by FACS. NIH 3T3 cells were cultured in high sugar DMEM medium and U937 in RPMI 1640 medium. UT7 cells which is cytokine-dependent cell line were grown in Iscove's modified Dulbeco's medium supplemented by GM-CSF. 3. Determination of viral titers: Retroviral vectors were transferred to packaging cell line 293. Retroviral containing supenatant was collected after transfection. The viral titers was tested on infection of NIH 3T3 cells by FACS analysis. 4. Western blot: Transduced CD41 +,UT7,U937 and MDA-MB-435 cells were analyzed by western blot to examine expression of transduced genes. RESULTS: A packaging cell line 293 produces high-titer MEK1 pMSCV retroviruses (3.1?10 7) and EC1-4 pMSCV retroviruses (1.0?10 8). With 8-folds dilution retroviruses,60.73% GFP positive cells have been obtained in MEK1 pMSCV transduced UT7 cells,72.56% in U937 cells and 30.57% in CD41 + cells,respectively. GFP positive cells have reached up 97.54 % in EC1-4 pMSCV transducted MDA-MB-435 cells. Phosphorylated MEK1 has been decreased in experiment group when TPO has stimulated CD41 + and UT7 cells or serum has stimulated U973 cells. This indicates that is a dominant negative effect of mutation MEK gene. EC1-4 gene transduced MDA-MB-435 cells have expressed EC1-4. CONCLUSION: The recombined murine stem cell retrovirus can effectively mediate gene transduction of CD41 +,UT7,U937 and MDA-MB-435 cells,and transduced genes can be stably expressed.