| HIM is the soil of orientation, proliferation, differentiation and development of HSC, of which hematopoietic stromal cell is an important component. Studies show that stromal cells which can be found in the organisms like bone marrow, spleen and thymus etc, are involved in the normal hematopoietic condition by producing HGF, ECM and the direct contact with HSC. The research and application on injury and repair of hematopoietic function in the past focus more on HSC, and it is needed to further study of the stromal cells.Umbilical cord blood has a large number of sources and is rich in HSC. There is a great promise for it's clinical application. The related systematic research is rather less in whether there exist hematopoietic stromal cells in HUCB, how about it's biological properties, and whether it can be expanded effectively in vitro? Accordingly, in this research, the umbilical cord blood CD34+ cells were cultured in Dexter system in order to investigate the possibilities of separating the stromal cells from HUCB and observe the growth of HUCBSC in different time and the proliferous effect of HGF and it's combination in vitro. Main results as follows:1. Dynamic observation on HUCBSC in culture: All umbilical cord blood samples were cultured with Dexter system. By 3-4d, the morphology of the cultured cells vared from round, triangle to irregularity; By 9-14d(at a median of 11.2d),stromal cell colonies formed and reached their maximum at 15-22d(mean 19.6d).With the period for culture elonged,cells be mainly composed of macrophage-like and fibroblast-like cell enlarged and grown up in round,ellipse and fusiform shape; by 28d, all culturer shown a significant increase in the number of adherent cell that filled the basis of petri dish.2. The morphology of HUCBSC: At the beginning of being cultured ,the shapes of the adherent cells display as small fusiform, triangle and sphere. With the prolong of culture time, these cells became bigger and bigger ,the cytoplasm color is Grayness-blue after Wright's staining and nucleus shape are sphere or ellips .The cells in spliting phases can be seen. Every one nucleus of the cells have one or two nucleoluses, which existing obviouslydifference with bone marrow stromal cells. Being observed from light microscope or invert microscope, these cells differentiate to three kinds of cells in 28d cultured: (1) Fibroblast liked cell, this cell's shape is fusiform with purplish red color cytoplasm, nucleus chromation is thick and including 1-3 nucleolus ;(2) Macrophage liked cell, these kinds of cells have big sphere soma, the interspace between cells have processus and gomphosising, nucleus chromatin is thick and including apparent nucleolus ;(3) Small-sphere cells with unknow nature, these kinds of small cells like lymphocyte, which have blue cytoplasm, nucleus chromatin is sligh and nucleolus is apparent. The ratio of these three kind of cells is 56.8%,38%,5.5%.3. Evaluation of HUCBSC: Cytochemical : the positive rate reached 100% in NSE stain and PAS stain, ALP stain for stromal cell also manifestes 35% positive, but in POX stain the result is negative. Immunohistochemistry stain: the positive rate of cord stromal cells for CD106, CD29, CD44, CD45, CD50, Fn, Lm , collagenâ…£ etc reached 96%, 93%,98%, 68%,72%,92%,74%,83% respectively.4. Ultrastructure of HUCBSC: There were rich mitochondrions and rough endoplasmic reticulums in cytoplasm of fibroblast. It has big nuclei without clear nucleolus,in which there were rich euchromatin and loosen heterochromatin;In macrophage cytoplasm, there were also rich mitochondrions and rough endoplasmic reticulums, in addition, Many Golgi body and Lysosomes vacuoles are easy to saw. It also has Ellipsoida nuclei with clear nucleolus,in which there were rich euchromatin and loosen heterochromatin tooï¼› As for the unknown little round cell, there were rich dissociation robosomes and mitocchondrials as well as rough endoplasmic reticulums and Golgi-bodies. Its nucli was irregular(some of them had nicks)including two or over ob... |
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