Research Of Aplastic Anemia Mechanism By Determining Soluble Stem Cell Factor And Its Receptor

Objective: To investigate the function of soluble stem cell factor (SCF) and its receptor (c-kit) in aplastic anemia (AA) , we chose the blood of AA children, the umbilical cord blood of normal mature infants and the plasma of health children as experiment materials, measured the stem cell factor receptor(c-kit) and the stem cell factor level of AA blood and umbilical cord blood by ELISA method and compared with that of health children.Methods: Take the blood of AA children, the umbilical cord blood of normal mature infants and the plasma of health children 3 to 5 ml for each sample, put in a tube with EDTA . Centrifugal the samples for 15 minutes at the rotate rate of 1500 rpm, then take the plasma and separate each into several parts according to the experimental procedure and then put them into a refrigerator and stored at -80℃ . Measure the stem cell factor receptor (c-kit) and the stem cell factor level by the method of ELISA. The intra of the c-kit reagent is less than 6% and the inter of it is less than 7.9%. The measuring sensitivity of c-kit reagent(DIACLONE, France) is 0. 095ng/ml, the liner range of the standard curve is 0.312-10.00ng/ml, R=0.98. The measuring sensitivity of SCF reagent(R&D, American) is 9.0pg/ml, the intra of it is less than 4% and the inter less than 10. 3%, the liner range of the log-log plot curve is 31. 2-2000pg/ml. All measure operation steps were strictly followed. The measure instrument is SIGAMA 960 made in German. Read the OD value of c-kit at the wave length of 450nm and the SCF at the wave length of 450nm and 630nm. ELISA assay was employed to determine the levels of soluble c-kit and SCF in bone marrow and peripheral blood of AA patients. Draw standard curves according standardOD value for further using.Results:The plasma c-kit level of health children is 16. 81±5.51ng/ml, that of AA is 13. 02±6.16ng/ml, of mature infant cord blood is 19.03±5.85ng/ml. There was significant difference between the levels of soluble c-kit of AA patients and health children(p<0. 01). There was significant difference between the levels of soluble c-kit of AA patients blood and mature infants cord blood (p<0.01) . There was no significant difference between the levels of soluble c-kit of health children blood and mature infant cord blood (p>0. 05).The plasma SCF level of health children is 468. 24±147. 21 pg/ml, that of AA is 363. 53±134.31pg/ml, of mature infant cord blood is 560.16±200.94pg/ml. There was significant difference between the SCF levels of AA patients and health children (p<0. 01). There was significant difference between the SCF levels of AA patients blood and mature infants cord blood (p<0. 01). There was no significant difference between the levels of soluble c-kit of health children blood and mature infant cord blood (p>0.05).Statistic analysis showed that there was no difference between the levels of bone marrow and the levels of peripheral blood. The levels of SCF in bone marrow and peripheral blood of AA patients are lower than controls.Conclusion: (1) The SCF level of AA blood is lower than that of health children, the lower levels of SCF in AA patients may contribute to decreased number of stromal cell. This shows that soluble c-kit may regulate properly SCF in AA patients. (2) The c-kit level of AA blood is lower than that of health children. (3) The SCF and c-kit levels of mature infants cord blood are both higher than that of health children. (4)The SCF and sc-kit levels in the plasma of the blood of AA children, the health children and mature infants cord blood are almost the same. It shows that sc-kit can regulate the growth potential of hemopoietic cell by combining with SCF or hold back its stimulation for hemopoiesis.