The Study Of BFGF And HGF Combined With VEGF-C On The Process That The Differentiation From MSCs Into LECs

BackgroundLymphedema is a chronically progressive disease that results in local tissue swelling due to reduced lymphatic transport capacity resulting in lymphatic fluid retention in the interstitial space.Lymphedema is divided into primary lymphedema and secondary lymphedema.Primary lymphedema is caused by genetic causes and other congenital causes of lymphatic vascular system injury.Secondary lymphedema is the result of surgery,radiotherapy,chemotherapy,filarial infection and other damage to the lymphatic vascular system.Lymphedema severely affects the physical and mental health of patients.The traditional treatment of lymphedema involves bundling therapy,manual lymphatic drainage,liposuction,etc.It relieves the symptoms to a certain extent and limits the progression of the disease,but it does not completely repair the damage to the lymphatic vessels.In recent years,the development of tissue engineering technology has provided new ideas for the treatment of lymphedema.It may be successful to cure lymphedema by means of cell transplantation to promote lymphangiogenesis and the reconstruction of lymphatic circulation.Mesenchymal stem cells(MSCs)can be used in many diseases with poor efficacy in traditional treatment methods because of their multidirectional differentiation potential,low toxicity,broad spectrum,and autologous transplantation.Bone marrow-derived mesenchymal stem cells(BMSCs)have better application prospects because they are easy to obtain,easy to isolate and proliferate rapidly.BMSCs can be induced differentiation into lymphatic endothelial cells(LECs)under certain conditions,which provide seed cells for lymphangiogenesis.In the process of differentiation of BMSCs into LECs,vascular endothelial growth factor c(VEGF-C)plays a central role and can be considered as a necessary factor.Vascular endothelial growth factor receptor 3(VEGFR-3)is a ligand of VEGF-C,and the binding energy of VEGF-C can accelerate lymphangiogenesis.Integrin ca9 can be directly bound to VEGF-C,which contributes to the formation of VEGF-C mediated lymphangiogenesis.Since integrin a9 and VEGFR-3 can bind directly to VEGF-C as ligands and exert corresponding physiological functions,and they are involved in lymphangiogenesis.There may be two signaling pathways,VEGF-C/VEGFR-3 and VEGF-C/Integrina9 in lymphangiogenesis.Basic fibroblast growth factor(bFGF)has a broad and strong biological activity,including promote cell mitosis,cell proliferation and cell differentiation.And it can be used as an inducer to induce differentiation of BMSCs into neuron-like cells,liver cells,vascular endothelium-like cells and so on.Therefore,it is reasonable to believe that the bFGF may successfully induce differentiation of BMSCs.Hepatocyte growth factor(HGF)can promote the proliferation of cells from various mesoderm and ectoderm and promote the migration of epithelial and endothelial cells.In addition,our previous study has demonstrated that HGF can be combined with VEGF-C to induce differentiation of BMSCs into LECs through upregulation of integrin a 9,it identify that HGF is a potent lymphangiogenesis factor.At present,the study of the process of inducing BMSCs to differentiate into LECs is still in its infancy stage,and there is no definite induced differentiation scheme,and the molecular mechanism of inducing differentiation is still not clear.In this experiment,multiple combinations of VEGF-C,bFGF and HGF as induction factors were used to explore the possibility of inducing differentiation of BMSCs into LECs,to find the optimal solution,and to explore the molecular mechanisms they are using to accomplish this process.To provide new ideas and experimental basis for the treatment of lymphedema by MSCs.PART Ⅰ BFGF,HGF AND VEGF-C WERE USED TO INDUCE BMSCS TO DIFFERENTIATE INTO LECSObjectiveTo observe the changes of cell morphology,to detect the expression of LECs markers and to investigate a more efficient method of inducing differentiation of BMSCs into LECs by using bFGF,HGF and VEGF-C.In the mean time,to determine the molecular mechanisms of lymphangiogenesis and to provide experimental foundation of experimental evidence of MSCs on the clinical treatment of lymphedema.Methods1)The expression of CD90,CD44 and CD34 in the BMSCs’ surface were assayed by flow cytometry(FCM);2)BMSCs were randomly divided into 8 groups:blank control group,VEGF-C group,HGF group,bFGF group,VEGF-C unite HGF group,VEGF-C unite bFGF group,HGF unite bFGF group and VEGF-C unite both HGF and bFGF group.After 10d culturing,the changes of cell morphology were observed by the inverted microscope;3)The relative expression level of LECs markers VEGFR-3,integrina9 and lymphatic vessel endothelial hyaluronan receptor 1(LYVE-1)were detected by western blot(WB)and real-time quantitative PCR(RT-qPCR).Results1)CD90 and CD44 were detected,CD34 was not detected in the BMSCs’ surface by FCM.2)BMSCs were long fusiform and fibroblast-like,and the cells in the VEGF-C group gradually became shorter and shorter,presenting a multilateral shape.3)The relative expression level of LECs markers were not expressed in blank control group,HGF group,bFGF group and HGF unite bFGF group by using both WB and RT-qPCR,which had the strongest expression in VEGF-C unite both HGF and bFGF group.Compared with VEGF-C unite HGF group and VEGF-C unite bFGF group,the expression of integrina9 in the former group was higher than the latter group(PWestern=0.002,PPCR=0.002),the expression of VEGFR-3 in the former group was lower than the latter group(PWestern=0.000,PPCR=0.000),and there were no significant differences in the LYVE-1 expression between the two groups(PWestern=0.208,PPCR=0.511).Conclusion1)The inducing method of VEGF-C+bFGF+HGF may be is the most efficient way to induce BMSCs to differentiate into LECs in this experiment.2)bFGF and HGF can only assist VEGF-C to induce BMSCs to differentiate into LECs.3)The differentiation which was induced by HGF may be promoted by stimulating VEGF-C/Integrina9 signaling pathway,at the same time,the differentiation which was induced by bFGF may be promoted by stimulating VEGF-C/VEGFR-3 signaling pathway.PART Ⅱ THE INTERACTION OF BFGF AND HGF IN DIFFERENTIATIONObjectiveTo determine the interaction of VEGF-C/VEGFR-3 And VEGF-C/Integrina9 signaling pathways in the differentiation of BMSC into LECs.VEGF-C as a basic inducer,bFGF and HGF as auxiliary inducers were used to co-induce BMSCs to differentiate into LECs,and change the sequence and interval time of bFGF and HGF.To determine the success of the differentiation from BMSCs into LECs by detecting the expression of VEGFR-3,LYVE-1 and integrina9.Methods1)BMSCs were randomly divided into 4 groups:blank control group,VEGF-C unite HGF+bFGF group(add HGF,VEGF-C and then add bFGF after 6 hours),VEGF-C unite bFGF+HGF group(add bFGF,VEGF-C and then add HGF after 6 hours),VEGF-C unite HGF and bFGF group(add HGF,VEGF-C and bFGF at the same time).After 10d culturing,the changes of cell morphology were observed by the inverted microscope.2)To set the time interval of 12h and 24h respectively,repeat the above steps.3)The relative expression level of LYVE-1,VEGFR-3 and integrina9 were detected by WB.Results1)The relative expression level of LYVE-1,VEGFR-3 and integrina9 were not expressed in blank control group.2)On the premise of the same time interval,LYVE-1,VEGFR-3 and integrin a9 were positively expressed in the other three experimental groups,and there were no significant differences in expression levels(P＞0.05).3)After changing the time interval between HGF and bFGF,the positive expressions of LYVE-1,VEGFR-3,and integrin a9 in the three experimental groups was still no significant difference.ConclusionOn the premise of the existence of VEGF-C,the roles of HGF and bFGF in inducing the differentiation of BMSCs into LECs were not in sequence.This shows that in the process of inducing differentiation,maybe HGF and bFGF are independent of each other.