Abstract:

To investigate the differentiation and expansion of megakaryocytes from CD34+ cells of umbilical cord blood and observe the change of their specific gene expression. MethodsCD34+ cells were obtained by  the immunomagnetic bead method and cultured in serumfree and stromafree media containing the following three cytokine combinations: thrombopoietin (TPO) +stem cell factor(SCF)+interleukin(IL)3+IL6,TPO+SCF+IL3 and TPO+SCF. The cells were collected on the 3rd, 7th, 10th and 14th day and expression of cell surface molecules was determined by fluorescence microscopy, and then cell apoptosis was determined by flow cytometery(FCM), maturation evaluation of the Mk ploidy was also carried out, and specific gene expression was determined by real time fluorescent quantitation PCR. ResultsIn vitro the CD34+ cells were effectively expanded. With the increase of time, expressions of CD41+, CD42b+, and CD61+ cells did not significantly change, but the optimal expansion of the Mk progenitors(CD34+/CD41+) was observed at day 7. Degree of maturation of the Mk cells also increased by evaluating the DNA content. TPO+SCF+IL3+IL6 was the optimal expansion method. Realtime fluorescent quantitation PCR displayed that transcription factor GATA1, NFE2, TXS, GPIIb and HPRT reached high levels at the 7th day, but