| Objective: As a new resource of hematopoietic stem cells, the umbilical blood has been widely used in the fields of transplantation, biological immunology and gene therapy because of its unique advantage. However, the low collection volume of the umbilical blood can not meet the demand of the adult transplantation. Researchers have kept exploring methods to efficiently separate and expand the umbilical cord blood stem/progenitor cells. This project aimed to find out a simple, high-efficient and practical method to separate the umbilical blood stem/progenitor cells and further to expand the purified stem/progenitor cells in vitro, trying to test the ability of proliferation of the umbilical blood stem/progenitor cells and to find out the optimal time for application. At the same time, we also compared the serum containing method and serum free method in expanding the umbilical cord blood stem/progenitor cells, trying to explore the possibility of the clinical application of the serum free method to expand umbilical cord blood stem/progenitor cells.Methods: The umbilical blood cells were separated and cultured in vitro and the number of monocytes was counted. The number of CD34+ cells was counted using flow cytometry. The colony forming ability was tested using CFU-mix colony culture. The separating and expanding effects of the two methods on the umbilical blood stem/progenitor cells were tested. 1. Density gradient centrifugation was adopted to separate the umbilical cord blood with self-mixed separatory fluid with a density of 1.064g/L and the traditional lymphocyte separatory fluid with the density of 1.077g/L. The separating effects of the two separatory fluids were compared according to the above said indexes. 2. After the monocytes were separated from the umbilical cord blood using the two separatory fluids respectively, they were expanded in culture media with cytokines (IL-3,SCF,GM-CSF). The above indexes were tested every week to compare the proliferating and differentiating effects of the two fluids on the umbilical cord blood stem/progenitor cells. 3.The umbilical blood monocytes were separated by the 1.064g/L separatory fluid and expanded in serum free culture system and horse serum containing culture system for 3 weeks. The above indexes were tested to compare the expanding effects of the two methods.Results: 1. The number of CD34+cells and the production rate of CFU-mix in the 1.064g/L group were respectively 3.7 times and 1.63times higher than the 1.077g/L group. This result suggested that the umbilical blood cells separated by the 1.064g/L separatory fluid have a larger volume of hematopoietic stem/progenitor cells and a higher proliferating ability. The number of monocytes in the 1.064g/L group was 0.6 times of the 1.077g/L group, suggesting a higher hematopoietic stem/progenitor cells collection rate of the 1.064 g/L separatory fluid. 2. With the co-effects of the early cytokines of IL-3,SCF,GM-CSF, the number of monocytes, and CD34+ cells had increased 58times and,26 times respectively in the 1.064g/L group in the second week of culture, while in the 1.077g/L group, the number had increased 27 times and 23times respectively. The ability of proliferation of the cells in both groups became the highest in the second week and the number of monocytes, CD34+ cells and CFU-mix in the 1.064g/L group were respectively 2.2times, 4.2times and 1.7times higher than the 1.077g/L group in the second week of expansion. There was a significant difference between the two groups. It suggested that the proliferating effect of 1.064g/L group be significantly higher than the 1.077g/L group. 3.The expanding effect of both the serum free culture system and the serum containing culture system got to its peak value in the second week with the number of monocytes and CD34+ were 55times and 25.8times in the serum free group and58 times and 26 times in the serum containing group. There was no significant difference between these two groups(P>0.05). This suggested that the serum free system and serum...
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