Study On Design Of Stretch Equipment And The Effect Of Stretch On Proliferation Of Marrow Mesenchymal Stem Cells

Marrow mesenchymal stem cells (MSCs), which root in the mesoblast, have the potentiality of differentiating into many kinds of cells. MSCs exist in different genera, such as chick, rat, rabbit, dog, human being, and so on. And they exit in connective tissue and apparatus, especially in the bone marrow. MSCs can differentiate into osteoblast, chondroblast, adipoblast, tendonblast, myoblast, cardiomyoblast, neuron, etc, in different inducement conditions. For this reason, MSCs have drawn more and more attention, and be used in tissue engineering, wound healing, cell transplant and gene therapy with the development of tissue engineering and gene engineering. But the quantity of MSCs is very few in animal and human being. There is only one MSC in about 100 thousands mononuclear in marrow and the quantity will decrease with the age increase. Obviously, this quantity cannot fit to the demand of cell engineering. So it is very necessary and important to find one way to purify and expand MSCs in man-made circumstance for the research of biologic characteristics, inducement, differentiation, and transplant of MSCs. Now there are many factors in vitro that could affect the proliferation of MSCs, and mechanical stimulation is an importance way. In recent years, that is always one importance field of biologic mechanics, which is the effect of mechanical factor on the growth and develop of tissue and cells. And scientists have carried out many research on it, and found a lots of rules. Stretch is one effective way in mechanical factor, and it was reported that stretch based on the best condition could promote the proliferation of cells. There is no detailed report about the effect of stretch on proliferation of MSCs up to now, so we do some research on it. The methods and contents include that: First of all, a kind of stretch equipment was designed, and the parameters of stretch were confirmed, and some preliminary experiments were done. Secondly, MSCs were separated by centrifugation in Percoll solution from adult rat, and cultivated and identifed MSCs. Thirdly, the effect of stretch on proliferation of MSCs was investigated, MSCs were stimulated by stretch equipment in different conditions, then the best stimulative condition of this stretch equipment and the change of expression of c-fos gene were analyzed by MTT and RT-PCR technique. Results: 1. Design of stretch equipment A kind of stretch equipment was designed based on the theory of stretch and rounded membrane. This equipment is controlled by a SCM, displays by LED, and its parameters can be adjusted easily and the range is linear. The range of the frequency is from 0.1Hz to 1Hz, and the intensity is from 2% to 15%, and the time is be controlled by manipulator. In general it can fit to the demand of this experiment. 2. Isolation and cultivation of MSCs Mononuclear cells were separated by centrifugation in Percoll solution (density 1.073g/mL). Then they were seeded in DMEM-LG supplemented with 10% fetal calf-serum and 1% antibiotic-antimyotic solution at a concentration of 1 ×106 cells/cm2。Cells were maintained at 37℃in a humidified atmosphere camber containing 5% CO2. The medium was changed after 72h, nonadhersive cells were removed with changing of medium, and twice weekly thereafter. When the cellss reached nearly 90%confluence. Then cell surface antigens were detected, and the result shown that CD34 was negative while CD44 and Fibronectin and CollagenI 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.. 3. Effect of stretch on proliferation of MSCs MSCs were stimulated by stretch with different time and different intensity while the frequency maintained 1Hz. It found that proliferation of MSCs could be regulated through stretch, and the effect of the stimulative time is more effective than that of stimulative intensity. The best condition is that the time is 60min, the intensity is 8%, and the frequency is 1Hz, then expression of c-fos gene in MSCs increased obviously. Experimental results suggested that the equipment that I designed could fit to the demand for my research, and MSCs could be achieved by centrifugation with Percoll solution, and proliferation of MSCs would be promoted by stimulation of stretch.