Effect Of Extracellular Matrix On Proliferation Of Rat Marrow Mesenchymal Stem Cells

Marrow mesenchymal stem cells (MSCs), which root in the mesoblast, have the potentiality of differentiating into many kinds of cells and strong ability of proliferation. In different inducement conditions, MSCs can differentiate into osteoblast, chondroblast, adipoblast, tendonblast, myoblast, cardiomyoblast, neuron, etc. Exogenous gene is easy to be inverted and expressed. So MSCs are the potential target cells which are used as gene therapy. For these characteristics, MSCs have drawn more and more attention, and be used in tissue engineering, wound healing, cell transplant and gene therapy, and so on. But there is not a standard and consistent method for isolation and cultivation and appraisal of MSCs up to now. Some methods are very cockamamie and it is not advantageous for the extending and standardization of MSCs. While MSCs proliferate slowly in a man-made circumstance, and may differentiate into osteoblast naturally. For these reasons, which referred before, it is very necessary to find out how to cultivate and expand MSCs in the man-made circumstance. It was reported that extracellular matrix (ECM) is one kind of thawless tissue, and it is not only the support tissue of cell, but also the lieu of supplying nutrition and expressing immunity. It adjusts the process of fetation, and chooses the adherent and transfer of cells, and even plays a very importance role in curing hurt, and growing and proliferation and differentiating and metabolizing of cells, and occurring and transfer of tumour. For this mechanism, we put emphasis on investigation on MSCs in order to find one way to isolate and cultivate and expand MSCs in vitro. It includes isolation and cultivation and appraisal of MSCs, and stimulating MSCs by ECM and detection of the result of stimulation. Methods: Mononuclear cells were separated by centrifugation with percoll solution (density 1.073g/mL). Then they were seeded in LG-DMEM supplemented with 15% fetal bovine serum and 1% antibiotic-antimyotic solution at a concentration of 1×10⁶ cells/cm², and cultured in a humidified atmosphere incubator at 37℃, containing 5% CO₂. The medium was changed after 72 hours, non-adhasive cells were removed, and medium changing will be implemented twice weekly thereafter. And relative biologic characteristics of MSCs were studied, including HE pigmentation,growth curve,cell cycle and ultrastructure. The cell surface antigens was detected, including CD34,Collagen I,CD44 and Fibronectin. And the cells were induced to differentiate into osteoblast, and AKP activity was detected by modified Gomori Calcium-Cobalt Method. Then MSCs were stimulated by ECM(Collagen I, Elastin), and the effect was analyzed by method of MTT to find out the best stimulative condition. Further more, analyzed expression of c-fos gene in MSCs after stimulation were investigated. Results: 1. The cell surface antigens were detected and the results shown that CD34 and Collagen I was negative while CD44 and Fibronectin and were positive. G0/G1 phase of cell cycle was 93.7%, and suggested that most of cells are not in period of proliferation. Analyse of AKP activity proved that these cells could differentiate into osteoblast. All of above results proved that the cells which we isolated and cultured in this experiment are MSCs. 2. MSCs were stimulated by different concentration of Collagen I, and the results showed that MSCs proliferated within 72h.The best stimulative conditions were 48h and the concentration of 20mg/mL Collagen I. 3. MSCs were stimulated by different concentration of Elastin, and the results showed that proliferous ratio decreased within 24h, and increased from 24h to 48h, but there was no obvious difference when compared it with control, and the proliferous ratio was restrain after 72h. It suggested that the effect of Elastin on proliferation of MSCs is not effective. 4. MSCs were stimulated by Collagen I of 20mg/mL concentration. It found that expression of c-fos gene increased obviously.