Study Of The Effect Of Supernatant Fluid Of Tumor Cells On Function And Activity Of Endothelial Progenitor Cells Derived From Rat Bone Marrow And The Effect Of PI3K/Akt In Endothelial Progenitor Cells Differentiation

IntroductionEndothelial progenitor cells(EPCs) are the precursor of endothelial cells,which can differentiate into endothelial cell(ECs) and have not express the mature ECs phenotype.The main source for the isolation of postnatal EPCs is the bone marrow. Sometimes EPCs can be mobilized to the peripheral blood and home to sites of target tissue to participate in the physiological or/and pathological vasculogenesis.There are two forms of new blood vessel formation:angiogenesis,the process of branching and elongation of existing vessels by migration and proliferation of resident mature endothelial cells;vasculogenesis,which is de novo vessel formation by in situ incorporation,differentiation,migration,and/or proliferation of BM-derived EPCs and hematopoietic stem cell(HSC).Increasing evidences suggest that malignant tumors may exist not only angiogenesis,but also vasculogenesis.These findings demonstrate that EPCs are quite important and critical to tumor neovascularization.The differentiation of EPCs may accommodate the expression of internal genes through complex signal transduction pathway and eventually cause EPCs to differentiate to mature ECs.At present the studies of EPCs differentiation are focus on the morphology and cell surface markers.It is difficult to separate EPCs from the other cells in morphology and the main approach relies on the molecular markers.AC133 is a new stem cell marker.It does not express in the mature ECs,and is thought of the most important marker to distinguish the ECs from EPCs.vWF(von Willebrand factor) exists in the Weibel-Palade bodies of ECs.The majority of vWF is secreted by ECs in plasma,which is a molecular marker of vascular endothelium.The studies show that the purified AC133⁺cells in vitro can differentiate into ECs.During the differentiation, EPCs obviously do not express AC133,and express the specific surface markers of mature ECs,such as vWF.PI3K/Akt is an important signal pathway which can regulate various cell growth, metabolism and differentiation.Phosphatidylinositol-3(hydroxyl) kinase(PI3K) is a significant member of growth factor receptor superfamily in the process of signal transduction,may be activated by a variety of cytokine factors.Serine/threonine protein kinase B(Akt) is a major kinase,located in the downstream of PI3K.PI3K and Akt are both key elements to promote cell survival and keep normal function.They constitute a cell signal transduction chain to promote cell growth and inhibit apoptosis during the process of stress reaction.Methods1,The separation,culture,purifies and identification of EPCsUse the Ficoll density gradient centrifuge combined with difference-speed adherence screening method to separate EPCs from rat bone marrow.Collect EPCs cultured 5 day,identify with laser confocal microscopy and the cells that AC133 and vWF are both positive are the differentiating EPCs.2,EPCs proliferation assayDigest and collect EPCs;Put cells into the 96 well cell culture cluster by 5×10³ and 200μl per well,37℃,5%CO₂ incubation 1-2h;After the cells fusion,non-blood serum DMEM to affect 12h;Add the tumor cells supernatant with different volume fraction (0%,10%,20%,40%);When detected,add 20μl MTT(5mg/ml) per well,incubation 4h, then DMSO 150μl per well;Shake 10min in the micro oscillator;Measure the OD value in wave length 490nm. 3,EPCs migration assayTake 5×10⁴ cells per chamber(volume 100μl) into Transwell upper chamber;Put 600μl supernatant fluid of tumor cells with different volume fraction(0%,10%,20%, 40%) into Tanswell lower chamber;When detected,flush three times with PBS;Then wipe the null migration cells with the cotton swab;Fix membrane superficies inferia with 100%methyl alcohol,haematoxylin dyeing;Cut the membrane,mount with the neutral gum;Count the cell number with microscope.4,EPCs Matrigel assayMove 5×10⁵ cells per well into 24 well cell culture cluster coated with Matrigel; After 24h incubation,take supernatant fluid of tumor cells with different volume fraction(0%,10%,20%,40%) into culture cluster;Count the number of the tube formation.5,Western BlotExtract the protein from cells;Measure the sample A₆₅₀ value;Polyacrylamide gel electrophoresis;Western Blot;Analysis.6,RT-PCRPrimer design;Extract total RNA from cells with TrIzol;Reverse transcription reaction;PCR.7,To exam the expression of EPCs differentiation factors after affected by LY294002Add LY294002,the specific inhibitor of PI3K/Akt at cultured day 3,day 7 and day 10 of EPCs.The last concentration of LY294002 is 20μmol/L.After affected 12h,test the mRNA expressions of AC133 and vWF by RT-PCR and the protein expression of p-Akt by Western blot,respectively.Results1,Cultivation and identification of EPCs After 24h original generation cultured,the cells are small and circular majority; after 1d,a few cells adhere;after 3d,some cells extend to spindle-shaped;at 5^th day, there are cell colonies;after 7d,the spindle-shaped cells continue to adhere and grow well;at 14d,the cells fuse to the monolayer like endothelial cell as the paving-stone shape.The cells that AC133 and vWF are both positive are the differentiating EPCs.2,The effect of supernatant fluid of tumor cells on proliferation of EPCsMake supernatant fluid of tumor cells with different volume fraction affect EPCs.The effect is the strongest when the volume fraction is 40%(P＜0.01).During 0-48h, the effect of supernatant fluid of tumor cells on proliferation of EPCs was stronger as the time expanded,and it has the dose-time dependence.3,The effect of supernatant fluid of tumor cells on migration of EPCsThe supernatant fluid of tumor cells may enhance the migration ability of EPCs.The effect reinforces at 6h(P＜0.01),reaches peak at 24h(P＜0.01) and drops at 48h(P＜0.01) which was still higher than that of the control.The effect is the most remarkable when the volume fraction is 40%.4,The effect of supernatant fluid of tumor cells on tube formation of EPCsThe supernatant fluid of tumor cells may enhance the tube formation ability of EPCs.When the volume fraction is 40%,the effect is the most remarkable and the tubes are more complex than the control.5,The protein expression of AC133,vWF,PI3K and Akt during the EPCs differentiationTest the expression of AC133,vWF,PI3K and Akt during the EPCs differentiation(at day 0,day 3,day 7,day 10 and day 14) by Western blot.The expression of AC133 is the strongest at day 0,and weaker at day 3.It is none at day 7, day 10 and day 14(P＜0.05).The expression of vWF does not change.The expressions of PI3K and Akt are the strongest at day 0,and weaker at day 3.The cultured time longer,the expression weaker.6,The mRNA expression of AC133,vWF,PI3K and Akt during the EPCs differentiationTest the mRNA expression of AC133,vWF,PI3K and Akt during the EPCs differentiation(at day 0,day 3,day 7,day 10 and day 14) by RT-PCR.The expression of AC133 is the strongest at day 0,and weaker at day 3.It is none at day 7,day 10 and day 14(P＜0.05).The expression of vWF does not change.The expressions of PI3K and Akt are the strongest at day 0,and weaker at day 3.The expression is much weaker accompanied by the cultured time elongation.7,The expression of factors in EPCs after LY294002 addedAdd LY294002 at cultured day 3,day 7,day 10 of EPCs.The last concentration of LY294002 is 20μmol/L.After affected 12h,test the mRNA expressions of AC133 and vWF by RT-PCR and the protein expression of p-Akt by Western blot,respectively.The results reveal that the mRNA expression level of AC133 at day 7 and day 10 is much lower than that at day 3(P＜0.05).The expression of vWF has no change(P＞0.05).The expression of p-Akt decreased gradually.It is P＜0.05,compared day 3 with day 10.Conclusions1,The supernatant fluid of tumor cells may enhance the proliferation,migration and tube formation abilities of EPCs.2,The abilities of EPCs on proliferation,migration and tube formation are related with the volume fraction and the affected time of the supernatant fluid of tumor cells.It is the most remarkable at volume fraction 40%and affected time 24h.3,The method of Ficoll density gradient centrifuge combined with difference-speed adherence screening method may separate EPCs from rat bone marrow. The purity and the survival percentage of EPCs are higher by this method.4,PI3K/Akt may possibly participate in the differentiation process from EPCs to ECs.5,The expressions of EPCs differentiation factors change after inhibitor LY294002 added,which presumes that the PI3K/Akt signal conduction pathway may be related with EPCs differentiation.This result may offer a route for the therapy of antivasculogenesis in tumor.