| ObjectiveIn order to investigate the hematopoietic function of placenta and study the effect of human placental chorionic tissue in different periods on proliferation of hematopoietic stem cells in vitro, further to know the changes of the hematopoietic function of placenta with the time.Looking out the method of economy and applicability to amplificate hematopoietic stem cells in vitro, If experiments can prove that the suspension of placenta can support the expansion of hematopoietic stem cells in vitro, so we can make use of the waste Saving a lot of expensive cytokines to amplificate hematopoietic stem cells, further used in the clinic successfully.Methods1. Collecting placenta villi tissue in early pregnancy(6to8weeks), mid-term (16and22weeks), the full-term placenta villi tissue (37weeks).2. Apply lymphocyte separation medium to sort out single nuclear cells, the sorting of CD34+cells is through flow cytometry.3. Set up four groups:early placenta (group B), mid-term placenta (group C), full-term placenta (group D), and black group (group A). Respectively amplification hematopoietic stem cells in co-culture way and research the colony formation ability after the expansion.4. Statistical analysis:All the data was performed using SPSS17.0statistical software. Results were expressed as mean±standard deviation(SD).the method of Single factor analysis of variance was used to analysis data. The level of significance was set to P<0.05.Results1. Separating CD34+cells from umbilical cord blood. The separated CD34+cells labeled with anti-human CD34FITC, the first shows the different group of cells, then grab positive cells from p5which was the scope that contains most of CD34+cells. Purity of CD34+cells was nearly99%through sorting by flow cytometry, the collection efficiency was95%.2. The results show that compared to initial concentration, the CD34+cells cultured with full-term placenta were amplified by (2.60±0.20) times than initial concentration, which was significantly higher than those of cells cultured with mid-term placenta (1.74±0.24) times and early placenta (1.14±0.12) times, but black group was reduction without amplification.3. Positive rate of CD34+cells after expansion by flow cytometry. Group A and Group B were all in low positive rate, but the positive rates of Group C and Group D were significantly higher, among them Group C has the highest positive rate about53.4%.4. Colony cultured in14days later, the results show the colony number of group C and group D were significantly higher than those of group A and group B. Among them the number of CFU-GM, CFU-GEMM, BFU-E of group C all a litter more than group D.Conclusion1. It is concluded that human placental extract in different period all can proliferate hematopoietic stem cells without any exogenous cell factors, this ability become more and more strong as time goes on.2. The colony formation ability of the amplified cells appears weakened after the first increase, this ability of the amplified cells in group C was the most strong slightly stronger than that of group D.3. In the later period of fetal development, the hematopoietic function of Placenta is gradually promoting the stem cells to differentiation and maturation. but the ability of prompting hematopoietic cell proliferation was not reduced. |
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