Conditioned Medium From Rat Bone Marrow Derived Mesenchymal Stem Cells Promotes The Proliferation Of Tenocytes Through ERK1/2

Marrow mesenchymal stem cells(MSCs) is a kind of non-hematopoietic stem cells, which can be easily obtained from diverse sources. MSCs has the capability of plasticity and can differentiate into many different types of cells, such as osteoblast and stromal cells. MSCs can participate in the wound healing by differentiation, which makes them become a prospective cell type in clinical. Recent researches showed that MSCs not only regenerate the damaged tissues by differentiation, but also by paracrine.Tendinopathy is one of common diseases in clinic. Based on the stem cells, tendon wound healing studies showed that many factors can induce stem cells directly differente into the tenocytes.In the area of stem cell therapy, many methods have been developed to obtain tenogenic differentiation from stem cells, such as cytokines, mechanical stretch, co-culture and so on. However, even if evidences showed that MSCs improve healing by paracrine, the paracrine effect of MSCs on the healing of damaged tendon remains unclear.The main purpose of this research is to explore the effect of the conditioned medium from rat marrow derived mesenchymal stem cell(r MSCs-CM) on the proliferation of tenocytes and the role of ERK1/2 in this process. The research results are as follows: ①Isolation and culture of r MSCs and tenocytesIsolation and culture of rat bone marrow derived MSCs(r MSCs): r MSCs are extracted from the bone marrow by whole bone marrow adherent culture method. Under the inverted phase contrast microscope, the morphological of r MSCs cells are observed to be uniform and swirl distribution, spindle or triangle, with obviously nucleus mass. Using flow cytometry to analyze the expression of cell surface antigens, the results showed that 92.9% of cells expressed CD44 and 96.9% of cells expressed CD90, while only 1.13% expressed CD34, which is consistent to the basic characteristics of MSCs.Isolation and culture of tenocytes: tissue pieces culture method was used to extract the tenocytes from the SD rats. Tenocytes are subcultured after 20 days. The cultured tenocytes exhibit long fibrous fusiform morphology, strong refraction and with obviously nucleus. ②The effect of r MSCs-CM on the proliferation of tenocytesBased on our previous results that there is not much differences between tenocytes treated with r MSCs-CM for 24 h, 48 h and 72 h, this thesis only studied the effect of r MSCs-CM on the proliferation of tenocytes after the treatment of 24 h. Cell counting is used to evaluate the proliferation of tenocytes in response to r MSCs-CM. The results showed that proliferation of tenocytes in r MSCs-CM-treated group is significantly higher than the control group, which is about 1.5 times compared to the control group(p<0.01), as well as the cell cycle results, which showed that the proliferation index of tenocytes in r MSCs-CM-treated group is significantly higher than the control group, suggesting that r MSCs-CM promotes the proliferation of tenocytes.③ r MSCs-CM promoted the proliferation of tenocytes through ERK1/2Western blot showed that expression of p-ERK1/2 was increased by the treatment of r MSCs-CM for 15, 30, 60, 90 and 120 min and the treatment for 15 min achieved 1.8 folds increase of control group(p<0.01), which indicated that r MSCs-CM may promote the proliferation of tenocytes through the activation of ERK1/2.We found that 50 μM of PD98059, an inhibitor of ERK1/2, abolished the r MSCs-CM-induced ERK1/2 activation in tenocytes while the r MSCs-CM-increased tenocyte proliferation was abolished as well. At the same time, the proliferation index of tenocytes in r MSCs-CM-treated group also reduced to control group. These results suggested that r MSCs-CM promoted the proliferation of tenocytes through the activation of ERK1/2.In conclusion, r MSCs-CM promoted the proliferation of tenocytes by influencing the cell cycle through ERK1/2 pathway.