The Effects Of PDGF-BB On The Proliferation Of Megakaryocytes And The TPO Production Of Bone Marrow Stromal Cells

Platelet-derived growth factor(PDGF)is a polypeptide growth factor which is stored in human platelet alpha granules.The PDGF family includes five different dimers,PDGF-AA,AB,BB,CC and DD,which play a biological role by binding PDGFa receptor and ? receptor.PDGFR is a transmembrane glycoprotein with tyrosine protein kinase activity.It consists of a and ? protein tyrosine kinase subunits.PDGF-BB can bind to PDGFR-a and ? receptors with high affinity,which is more potent than other subtypes of PDGF.Studies have shown that PDGF-BB can promote the growth and differentiation of various connective tissue cells such as fibroblasts,endothelial cells and smooth muscle cells et al.PDGF-BB can also promote hematopoiesis,such as promoting erythropoiesis and multipotent hematopoietic progenitor cell proliferation.Michalvicz and Katz found that PDGF-BB play an important role in the formation of BFU-E and CFU-GEMM.However,the specific mechanism of PDGF-BB promoting hematopoiesis remains unknown.It may be regulated by bone marrow mesenchymal cells in the bone marrow microenvironment.Therefore,the aim of this study is to investigate the effect of PDGF-BB on the proliferation of CFU-MK in vitro and to compare it with IL-3,IL-6,TPO and GM-CSF.Further study the possible mechanism of PDGF on the growth of megakaryocyte:PDGF-BB can stimulates bone marrow mesenchymal cells produce TPO,which indirectly promotes hematopoiesis.MethodPart I PDGF-BB promotes megakaryocyte formationi.Effects of PDGF-BB on murine CFU-MK formation1.The formation of murine CFU-MK after treated with different concentrations of PDGF-BB(0 ng/ml,5 ng/ml,10 ng/ml,20 ng/ml,50 ng/ml,100 ng/ml)2.The effect of different cytokines(PDGF-BB,IL-3,IL-6,GM-CSF and TPO)on the formation of murine CFU-MK3.Synergistic effects of PDGF-BB,IL-3 and IL-6 on murine CFU-MK formation4.The effect of PDGF-BB on the formation of CFU-MK after adding anti-IL-6 antibody,anti-IL-3 antibody or anti-TPO antibody.ii.Effects of PDGF-BB on human CFU-MK formationThe effect of various concentration of PDGF-BB(0 ng/ml,5 ng/ml,20 ng/ml,50 ng/ml,100 ng/ml)on the formation of human CFU-MK.Part ? The effects of PDGF-BB on the TPO production of bone marrow stromal cells1.The effect of PDGF on the proliferation and anti-apoptosis of human MSCs was detected by CCK-8 and flow cytometry.2.The expression of PDGFR-? on MSCs was detected by Q-PCR and flow cytometry.3.The phosphorylation of ERK and STAT3 after treated with PDGF was detected by Western blot.4.The expression of TPO mRNA in MSCs was determinded by Q-PCR.ResultPart I PDGF-BB promotes megakaryocytes formationi.The effect of PDGF-BB on murine CFU-MK formation1.PDGF-BB promoted CFU-MKformation in dose-dependent manner and 50 ng/ml had the most obvious effect(n = 5,P<0.001).2.The effect of PDGF-BB on the formation of CFU-MK was higher than GM-CSF(P<0.05)),but weaker than IL-3,IL-6 and TPO(P<0.05);3.IL-3 + IL-6 had a significant synergistic effect on the proliferation of CFU-MK(n =5,P<0.00001);4.Compared with PDGF-BB group,the numbers of CFU-MK in PDGF-BB +IL-3 MoAb group had no significant difference(n = 4,P = 0.127);5.The numbers of CFU-MK in PDGF-BB group was twice as much as PDGF-BB +IL-6 MoAb group(n=4,P=0.0003);6.The numbers of CFU-MK in PDGF-BB group was twice as much as PDGF-BB +TPO MoAb group(n=4,P= 0.002);ii.Effects of PDGF-BB on human CFU-MK proliferation PDGF-BB promoted the formation of human CFU-MK in a dose-dependent manner and 50 ng/ml had the most obvious effect(n = 5,P<0.001).Part II PDGF stimulates bone marrow stromal cells produce TPO1.PDGF-BB promoted the proliferation of human MSCs and 50 ng/ml had the most obvious effect(n=8,P =0.0027).PDGF-BB reduced the proportion of total apoptotic cells significantly(n=5,P=0.00004).2.PDGFR-? was expressed higherly on human MSCs showed by flow cytometry and Q-PCR.3.After treated with PDGF-BB,the phosphorylation of ERK and STAT3 in human MSCs was increased showed by Western blot.4.The expression of MSCs TPO mRNA was significantly increased after treated with PDGF-BB(n=7,P=0.021).Cnclusion1.PDGF-BB can promote the CFU-MK formation.2.PDGF-BB has a pro-proliferative and anti-apoptotic effect on human MSCs;Human MSCs expressed PDGFR-P,PDGF-BB can activate MAPK/ERK and STAT pathway;3.PDGF-BB can promote the expression of MSCs TPO mRNA.