Effect Of Extracellular Matrix, Cytokine And Mechanical Stimulation On Proliferation Of Rat Marrow Mesenchymal Stem Cells

Stem cells are the special cells that have self-renewal and multi-differentiation ability. According to the different differentiation potential, stem cells can be divided into three types: totipotent stem cell (TSC), pluripotential stem cell (PSC) and unipotent stem cell (USC). Under some special conditions, bone marrow-derived mesenchymal stem cells (MSCs) can be induced to differentiate into osteoblasts, chondrocytes, muscle cells, adipocytes and so on. At the present, MSCs are studied and used widely and have become the main source of cells for clinical cell transplantation and tissue engineering. For the slow growth ability and limited life span of MSCs in vitro, it is the prerequisite for the research and clinical application to expand MSCs in vitro and to maintain its multi-differentiation ability.It has been proved that many physical and chemical factors play an important role on the proliferation of cells, such as extracellular matrix (ECM), cytokine and mechanical stimulation. Collagen I (Col I) is one of the main components of ECM. Basic fibroblast growth factor (bFGF) and appropriate mechanical stimulation can significantly promote the proliferation of kinds of cells. However, little is known about the effect of Col I, bFGF and mechanical stimulation on proliferation of MSCs. Thus, to find the appropriate physical and chemical stimulation conditions in promoting the proliferation of rat MSCs (rMSCs) in vitro, we focus on the effect of Col I, bFGF and mechanical stimulation on proliferation of rMSCs by using them alone and synergetically. The main research works and results are as follows:1. Isolation and culture of rMSCs in vitroThe rMSCs were isolated by centrifugation with 1.073 g/mL percoll solution. The results indicated the isolated cells were homogeneous histoleucocyte. Cultured in DMEM with 10% FBS for 24 hours, the cells adhered, divided and grew into colony. After about 2 weeks they achieved confluence, and showed fibroblast-like morphology. The nonadhesive cells were removed by changing the medium. After about 1 week, the subcultured spindly cells achieved confluence.2. Identification and cell cycle analysis of rMSCsNucleoli of cultured cells, stained by Gimsa, were round or ellipse. Several dark purple nucleoli were thus clear meanwhile cytoplasm was lilaceous. We detected that the surface antigen CD34 negative while CD44 and CD29 were positive, and G0/G1 phase cells were (89.74±3.87) %, S phase were (2.49±2.20) % and G2/M phase were (7.70±3.70) %.3. Effect of Col I, bFGF and the combined on proliferation of rMSCsThis paper measured the growth curve of rMSCs, and then investigated the effect of Col I and bFGF on proliferation of rMSCs by using them alone and synergetically. The result showed that: rMSCs growth latency was for a period of 1 to 2 days, and the growth period was from 3 to 7 days, after seven days cells turned into the period of growth platform. The proliferation of rMSCs could be significantly promoted by the 0-40μg/ml Col I, especially 20μg/ml Col I. Cells cultured 8 days with 20μg /mL Col I was the best stimulative conditions for rMSCs proliferation. Also the proliferation of rMSCs could be significantly promoted by 0-20 ng/ml bFGF, especially by 7 ng/ml bFGF, and the best stimulative conditions for cell proliferation were 8 days with 7 ng /mL bFGF. The effect of Col I and bFGF were significantly higher by using them synergetically than by using them alone. Col I and bFGF can promote synergetically the proliferation of MSCs.4. Effect of Mechanical strain and the co-effect of mechanical strain, Col I and bFGF on proliferation of rMSCsThis paper characters the effect of mechanical stretch on proliferation of rMSCs. The effect on proliferation of rMSCs by single axis mechanical stretch (5% and 10%, 1Hz, 15, 30 and 60minutes) was investigated. The result showed that: in contrast to control group, the proliferation of rMSCs could be promoted after exposure to 5% strain for 15 and 60 minutes, especially for 15 minutes. After strain duration time of 30 minutes, a lower proliferation rate was measured compared with control levels. The result of 10% strain on proliferation of rMSC was in similar to 5% strain and the effect was higher than 5% strain. So exposed to 1 Hz, 10% strain for 15 minutes was an appropriate strain condition for promoting proliferation of rMSC in vitro. Furthermore, we found that the effect of the co-effect of mechanical strain, Col I and bFGF on proliferation of rMSCs was the highest.The result is helpful to understand the various physical and chemical factors and the co-effect on the proliferation of rMSCs. It provides experimental basis and reference methodology for seeking to promote proliferation of MSCs in vitro.