ABSTRACT
To further explore the mechanism of congenital pyrimidine 5'-nuleotidase I (P5'N-I) deficiency, on the basis of purification of the protein, the molecular weight and amino acid composition were analysed by mass-spectrograph and amino-acid analyzer, microsequencing and bioinformation analysis of P5'N-I were performed after it was hydrolysed by trypsin. The results showed that three fractions were found in the purified P5'N-I and their molecular weights were 26,952.9, 55,476 and 110,938, respectively. The sequence from one of the peptide fragments was I-E-G-P-T-I-R-Q-I-E. The homologous sequence was not found after comparision with the ten-amino-acid sequence in GenBank by blast procedure. Amino acid analysis indicated that P5'N-I was composed of 18 amino acids at least, and 243 amino acid residues. In conclusion, the enzyme might be an allosteric enzyme, there might be homologous dimer or tetramer in physiological status of normal human erythrocyte, the microsequence could be designed as the probe for fishing the genes of interest. The composition of amino acid might be an important information in determination of its protein primary structure.
Subject(s)
Humans , 5'-Nucleotidase , Blood , Chemistry , Amino Acid Sequence , Amino Acids , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Erythrocytes , Mass Spectrometry , Molecular Weight , Peptide Fragments , Chemistry , Sequence Analysis, ProteinABSTRACT
To elucidate the role of fetal bone marrow stromal cells (FBMSC) in cooperation with exogenous cytokines in supporting the in vitro expansion of cord blood CD34(+) cells which were purified by negative immunomagnetic selection, FBMSCs were cultured with different combinations of cytokines including SCF, IL-3, IL-6, FL, G-CSF and EPO in a 14-day liquid culture system. The results showed FBMSC plus SCF, IL-3, IL-6, FL and EPO was the most effective combination which increased total nucleated cells, CFU-GM, BFU-E and CD34(+) cells by (692.4 +/- 52.7) fold, (237.1 +/- 106.6) fold, (114.8 +/- 32.8) fold and (25.3 +/- 10.1) fold, respectively. Our studies indicated that fetal bone marrow stromal cells combined with above-mentioned cytokines can efficiently expand cord blood CD34(+) cells.
ABSTRACT
Objective： To elucidate the role of bone marrow stromal cells in cooperation with exogenous cytokines in hematopoiesis. Methods： Fetal bone marrow stromal cells (FBMSC) was combined with cytokines including SCF,IL-3,IL-6,GM-CSF in a 5-day liquid culture system of adult bone marrow mononuclear cells, then we cultured bone marrow derived CD34+-enriched cells with FBMSC+SCF+IL-3+IL-6+G-CSF+EPO for 2 weeks. Results：FBMSC were in good cooperation with above mentioned exogenous cytokines. When CD34+-enriched cells from adult bone marrow were cultured with combinations of FBMSC, SCF, IL-3, IL-6, G-CSF and EPO, total nucleated cells, CFU-GM, BFU-E and CD34+ cells were increased by 119.6±30.9, 54.6±17.4, 25.2±4.4, 11.1±4.2 folds, respectively. Conclusion：FBMSC in cooperation with exogenous cytokines support the in vitro expansion of human hematopoietic progenitor cells efficiently.
ABSTRACT
Objective： To elucidate the role of bone marrow stromal cells in cooperation with exogenous cytokines in hematopoiesis. Methods： Fetal bone marrow stromal cells (FBMSC) was combined with cytokines including SCF,IL-3,IL-6,GM-CSF in a 5-day liquid culture system of adult bone marrow mononuclear cells, then we cultured bone marrow derived CD34+-enriched cells with FBMSC+SCF+IL-3+IL-6+G-CSF+EPO for 2 weeks. Results：FBMSC were in good cooperation with above mentioned exogenous cytokines. When CD34+-enriched cells from adult bone marrow were cultured with combinations of FBMSC, SCF, IL-3, IL-6, G-CSF and EPO, total nucleated cells, CFU-GM, BFU-E and CD34+ cells were increased by 119.6±30.9, 54.6±17.4, 25.2±4.4, 11.1±4.2 folds, respectively. Conclusion：FBMSC in cooperation with exogenous cytokines support the in vitro expansion of human hematopoietic progenitor cells efficiently.
ABSTRACT
For exploring pathogenesis of pyrimidine 5'-nucleotidase (P5'N) deficiency, a quantitative assay method for human erythrocyte pyrimidine 5'-nucleotidase was established. The specific substrate uridine monophosphate (UMP) of P5'N was used as ligand. The UPM-ADH-Sepharose 4B affinity column was prepared. P5'N of human erythrocyte was purified by ammonium sulfate fractionation and precipitation, ion chromatography and affinity chromatography. Rabbit anti-human P5'N antibody was acquired by immunizing rabbits with purified P5'N. Using rabbit anti-human antibody as the coated anti-body and HRP-rabbit anti-human antibody as demonstrated anti-body, the double antitody sandwich ELISA for quantitative assay of human erythrocyte P5'N was established after square rank trial, antigen blocked trial and antigen substituted test. Results showed that the titer of rabbit anti-human erythrocyte was 1:4 and the sensitivity of double antibody sandwich ELISA was 5 ng/ml. Its blocking rate was more than 95% and the rate of substitution less than 30%. The content of P5'N was (71.77 + 10.98) ng/mg NHP in normal human erythrocyte. A new ELISA method for quantitative determination of human erythrocyte P5'N was first established. It not only had high specificity and sensitivity but also could assay the minimun content of P5'N as 5 - 20 ng/ml. It could be a suitable method for large sample in clinics.