| Mesenchymal stem cell is a kind of adult stem cell, the multi-potential of these cells, their easy isolation and culture, as well as their high ex vivo expansive potential make these cells an attractive therapeutic tool. Cumulative data have been documented that mesenchymal stem cells can differentiate into osteoblasts, chondrocytes, adipocytes, tenocytes, myotubes, neural-like cells, hematopoietic-supporting stroma and other non-hematopoietic cells derived from mesoblast or ectoblast. MSCs should be isolated and purified easily in vitro, and expressing many objective genes in vivo. Recent years, MSCs have been focus in the fields of gene and tissue engineering.In this study, MSCs were isolated and purified, and it's biological features, osteoblastic differentiation in vitro and application in the fields of gene transfection were investigated by using the methods and technologies of cytobiology, experimental hematology, immunohistochemistry and molecular biology. The main results are as follows:1. The biological characteristics of human bone marrow mesenchymal stem cellsA culture of mesenchymal stem cells was initiated from bone marrow low-density mononuclear cells separated by ficoll centrifugation and plated at 5 X 105cells/dish. MSCs were spindle-like in morphology with filling flasm adhering to the dish. MSCs colonies were found after nine days of growth. Cells achieved confluence when the MSCs incubated for another two weeks.The growth curve, the doubling time of MSCs and the surface antigenic marker present on MSCs were investigated. The results showed that the doubling time of MSCs is 31.4h and MSCs were negative for CD45.2. The effects of several factors on the proliferation of human bone marrow mesenchymal stem cellsThe effects of different serum concentration, cell number, cytokines and condition-mediums on the proliferation of MSC were studies. The results showed asfollow:The colony number of MSCs was increased in 5%-30% concentration of serum in dose dependent, and was not further increased when the concentration of serum reach 40%.Two hundred cells or 500 cells were plated per dish contained DMEM supplemented 20% NBS. We picked 7 dishes each group and choosed 5 MSCs colonies randomly per dish to count the cells number of each colony, the results showed that in 200 cells/dish group ,there is no colony be found until 13 day, however MSCs became older, the cell number of each colony is 55.77±34.80; in 500 cells/dish group, colonies were found at 9 day, the cells number of each colony is 78.97 ± 43.04. The doubling time is 53.8h and 34.3h correspondingly.IL-6 (50ng ·ml-1), IL-3 (10U ·ml-1), bFGF (5ng·'ml-1) significant difference as compared with control (p<0.01) , IL-lα (10-8mol±1-1) elicited difference as compared with control (p<0.05 ) . They all promoted the proliferation of MSCs. INF-% (25ng·ml-1), GM-CSF (100ng·ml-1) å'Œ TGF- β (2ng·-ml-1) had no significant effect on the growth of MSCs . bFGF can promoted the proliferation of MSCs in 3ng ?ml-1 - 7ng ?ml-1 concentration in dose dependent and can significantly promoted the proliferation of MSCs (p<0.01) , 7ng ?ml-1 bFGF is better than 5ng ?ml-1 bFGF to promoted the proliferation of MSCs (p<0.05) .There are no longer promoted the proliferation of MSCs when the concentration of bFGF is beyond 7ng ?ml-1.Conditioned Medium from MSCs (MSC-CM) (10%) . The number of colonies was difference as compared with control (p<0.05); Conditioned Medium from buffalo rat liver cell (BRL-CM ) (10%) stimulated pure MSCs also.The number of colonies was significant difference as compared with control (p<0.01) .Both MSC-CM (10%) and BRL-CM (10%) can promoted the proliferation of MSCs. MSC-CM (10%) is better than BRL-CM (10%) to promoted the proliferation of MSCs (p<0.05) .The results suggest that MSCs might excrete some cytokines to promote itself proliferation. 3. osteoblastic differentiation in vitro of human bone marrow MSCsIn vitro the attached MSCs at passage 4 were treated with 1×10-8mol ?1-1... |
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