The Expression Of PDGFR? Regulated By BFGF During The Recovery Of Human Bone Marrow Mesenchymal Stem Cell After Irradiation And The Mechanisms Involved

Bone marrow is highly sensitive to radiation and hematopoietic damage is one of the main manifestations of radiation sickness.The function of bone marrow is slow to recover after irradiation and human bone marrow mesenchymal stem cell dysfunction is believed to be the key cause of this process.Human bone marrow mesenchymal stem cell dysfunction after irradiation is characterized by decreased proliferation and osteogenic differentiation.The number of osteoblasts and hematopoietic function activity are closely related to bone marrow hematopoietic function.Thus,the recovery of human bone marrow mesenchymal stem cell proliferation and osteogenic differentiation capacity is of great significance in repairing hematopoietic function of bone marrow.Basic fibroblast growth factor(bFGF)is a commonly occurring growth factor.Recent studies showed that bFGF increased the radiation-resistance of several types of stem cells;however,the effects of bFGF on human bone marrow mesenchymal stem cell which may be damaged by radiation still need to clarify.Platelet-derived growth factor receptor alpha(PFGFR-?)is a newly identified protein that is critical for human bone marrow mesenchymal stem cell proliferation and osteogenic differentiation.bFGF was suggested to be involved in the regulation of PDGFR-? expression.In this study,we evaluated the effects of bFGF on human bone marrow mesenchymal stem cell which was damaged by radiation and conducted a preliminary investigation the role of dynamic changes in PDGFR-? expression,thereby providing a theoretical basis for the clinical treatment of radiation sickness.Firstly,we cultured and identified the human bone marrow mesenchymal stem cell in vitro,then Cell Counting Kit-8(CCK-8),Flow cytometric analysis,Western blotting and Alizarin red staining assay were performed to investigate the effects of irradiation on human bone marrow mesenchymal stem cell,such as the cell proliferation,apoptosis,osteogenic differentiation after different dose of X ray irradiation(0Gy,6Gy and 12Gy).According to the above results,we screened the appropriate dose of irradiation.Secondly,the methods above which include CCK-8,Western blotting and Alizarin red staining assay were performed to investigate the effects of bFGF(0,1,5,10 and 20 ng/ml)on irradiated human bone marrow mesenchymal stem cell,such as the cell proliferation and osteogenic differentiation.According to the above results,we screened the appropriate concentration of bFGF for the following experiments.Lastly,Western blotting was performed to investigate the expression level of PDGFR? among three groups: non-irradiated human bone marrow mesenchymal stem cell,irradiated human bone marrow mesenchymal stem cell without bFGF treatment and irradiated human bone marrow mesenchymal stem cell with bFGF treatment.As a result,the cells were exhibited a spindle shape and a spiral growth pattern and tested positive for CD44,CD73,CD90 and CD105,negative for CD45,CD34 and HLA-DR.The positive rate of these indicators exceeded 99% and the negative rate was lower than 1%.After 21 days of osteogenic differentiation induction,orange-colored calcium nodules were observed by alizarin red staining,indicating that the cultured cells were human bone marrow mesenchymal stem cells.Radiation exposure significantly inhibited human bone marrow mesenchymal stem cell growth and osteogenesis in a dose-dependent manner(p<0.05).A significant inhibition of irradiated cell growth compared with non-irradiated cell growth was observed after 3 days(P < 0.05).After 7 days,significantly greater inhibition of 12 Gy treated cell proliferation was observed compared with the 6 Gy treated cells(P < 0.05).The number of nodules were highest in the control(0 Gy group)and higher in the 6Gy group(P < 0.01 vs.control)and least in 12 Gy group(P < 0.001 vs.control).The apoptosis was only significant for cells exposed to high dose irradiation at 24-h time-point(p<0.05),and no difference was detected at the later time-point(p>0.05),6 Gy was the appropriate dose.Furthermore,bFGF improved the recovery of human bone marrow mesenchymal stem cell proliferation and osteogenic function impaired by irradiation with the dose of 6 Gy(P<0.05),but 20ng/ml bFGF did not promote human bone marrow mesenchymal stem cell proliferation that was damaged by irradiation(P>0.05).After 5 days of bFGF treatment,cultures containing 1,5,and 10 ng/ml bFGF exhibited significantly higher cell counts than those without bFGF(P < 0.05).At 7-day time-point,cultures containing 5 ng/ml bFGF exhibited higher cell counts than the other bFGF-treated cultures,with the most significant differences observed after 9 days in culture(P < 0.05),there was no significant difference in proliferation between the cultures treated with 20 ng/ml bFGF and those without bFGF treatment.bFGF at 5 ng/ml enhanced proliferation to a greater extent than bFGF at 1 ng/ml or 10 ng/ml and Alizarin red staining showed that the calcium nodule count in irradiated human bone marrow mesenchymal stem cell with bFGF treatment was significantly increased compared to the irradiated human bone marrow mesenchymal stem cell without bFGF treatment.Moreover,the expression level of PDGFR? matched with the proliferation curve of human bone marrow mesenchymal stem cell that was treated with irradiation and bFGF.The level of PDGFR? was highest in non-irradiated cells,higher in irradiated human bone marrow mesenchymal stem cell that with bFGF treatment and lowest in irradiation cells that without bFGF treatment.According to the results above,we concluded that: First of all,irradiation impairs human bone marrow mesenchymal stem cell proliferation and osteogenic differentiation remarkably,but there was insignificant influence on apoptosis.Second,bFGF improves the recovery of human bone marrow mesenchymal stem cell proliferation and osteogenic function impaired by irradiation and 5 ng/ml was identified as the optimal bFGF concentration.Third,the mechanism by which irradiation impairs human bone marrow mesenchymal stem cell involves decreased PDGFR? expression and increased PDGFR? expression involves in repairing effect of bFGF on irradiation damage of human bone marrow mesenchymal stem cell.