Effect Of β-elemene On The Activity And VEGF Expression Of Rat Bone Marrow Derived Endothelial Progenitor Cells Induced By Tumor Cells Supernatant

Endothelial progenitor cells (EPCs) are the precursor of endothelial cells in the embryonic development period, which can differentiate into endothelial cells (ECs). To be born, EPCs mainly in the morrow.In some cases EPCs can be mobilized to peripheral blood and homed to the target tissue participating the physiological or pathological angiogenesis. Endogenous factors like ischemia,hypoxia,damage,stress and extrinsic factors as cell factors VEGF,GM-CSF,G-CSF,drugs can promote the mobilization of EPCs.After VEGF injeceted in the mice,the EPCs of circulating blood increase generously,the ability of generation and migration also increased.The researchers discover that EPCs play an important role in many ways as ischemia disease,wound healing,tumor angiogenesis.The elementary process is EPCs mobilized and migrated to peripheral blood,homing and recruitment to ischemia tissues,differented to endothelial cells.The growth of entity tumor depended on tumor neovascularity. Inhibiting neovascularization to restrain the development of tumou will become a new important target and method.In the past,the researchers consider that the migration and generation of tumor local endothelial cells to form the tumor vessel.But the therapy of anti-tumor angiogenesis to aim at endothelial cells of tumor vessel is not very good. It also can not explan tumor angiogenesis mimicry and so on very well.Now the reseachers discover that EPCs play a great role in the tumor angiogenesis. EPCs may become another antineoplaston target.Elemene extracted in the traditional Chinese medicine curcuma aromatica salisb (our country's unique new kind names radix curcumae) aetherolea is the active component of anticancer.β-elemene is the main active component of elemene.The specific study about the main mechanism ofβ-elemene is in the primary phase.The in vitro study demonstrate thatβ-elemene can arrest many tumor cells from S to G2/M phase,inhibit their proliferation,down regulate proteinum expression of VEGF-C,VEGFR-3,β-EGF of adenocainoma of lung and throat squamous cell carcinoma,depress the microvessel density.This research purpose is studying the effect ofβ-elemene on the EPCs induced by tumor cells supernatant and whether can inhibit EPCs in the tumor vasculogenesis.Method一.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 4 day, identify with laser confocal microscopy and the cells that AC133 and vWF are both positive are the differentiating EPCs.二.MTT assay EPCs proliferationDigest 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 40% volume fraction tumor cells supernatant,after 12h add different densityβ-elemene Oμg/ml,5μg/ml, 10μg/ml,20μg/ml;after 48h 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.三.Matrigel assay the vasculogenesis activity of EPCsDigest and collect EPCs; Put cells into the 24 well cell culture cluster by 5×103 per well; Add 40% volume fraction tumor cells supernatant,after 12h add different densityβ-elemene 0μg/ml,5μg/ml, 10μg/ml,20μg/ml,to continue culture 24h.Observe the number of the tube formation,choosing 5 field of vision per well random and getting the average value.四.Flow cytommetry detect EPCs cell cycleSelect 5d EPCs; Add 40% volume fraction tumor cells supernatant,after 12h add different densityβ-elemene 0μg/ml,5μg/ml, 10μg/ml,20μg/ml to continue culture 48h.Trypsinizate EPCs and make it into ventrifuge tube,1000s/min,centrifuge 10min,PBS washing two times. Abscising supernatant, fix it with 4℃precooling 75% alcohol,4℃overnight.1000s/min,10min,PBS wash one time,abscise supernatant,and add 200μg/ml RNase A,37℃incubating one hour; Add 20μg/ml PI,protected from light 30min 4℃.Flow cytomertry analysis EPCs cell cycle.五. Annexin-V/FITC analysis EPCs apoptosisSelect 5d EPCs;Add 40% volume fraction tumor cells supernatant;After 12h add different density (3-elemene Oμg/ml,5μg/ml,10μg/ml,20μg/ml to continue culture 48h.Trypsinizate EPCs and make it into ventrifuge tube,1000s/min,centrifuge 10min,PBS washing two times, abscising supernatant.Differently add FITC and Annexin-V according to kit ditection,incubation 10min and flow cytomertry analysis.六.RT-PCR analysis mRNA expression of EPCs VEGF and VEGFR-2Extract every group EPCs totol RNA with Trizol,according to kit ditection reverse transcription reaction;PCR.七. Statisticsl AnalysisUse SPSS13.0 software to carry out statistical analysis.Experimental result use ANOVA analysis.P<0.05 means the difference to be significant.Result八.Cultivation and identification of EPCsAfter 24h original generation cultured, the cells are small and circular majority; after Id, a few cells adhere; after 3d, some cells extend to spindle-shaped; at 5th 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 AC 133 and vWF are both positive are the differentiating of EPCs.二.The effect ofβ-elemene on the proliferation of EPCs induced by tumor cells supernatantAffter different density(5,10,20μg/ml)β-elemene affect EPCs 48h and 20μg/mlβ-elemene affect EPCs different time(12,24,48h). Compare to the control, the proliferation of EPCs is depressed obviously (P＜0.01)三. The effect of p-elemene on the activity of vasculogenesis of EPCs induced by tumor cells supernatantAffter different density(5,10,20μg/ml)β-elemene affect EPCs 48h and 20μg/mlβ-elemene affect EPCs different time(12,24,48h). Compare to the control,the tube formation of EPCs is depressed obviously (P＜0.01)四. The effect of P-elemene on the apoptosis of EPCs induced by tumor cells supernatantWhen the density of P-elemene is 5μg/ml, the apoptosis of EPCs changes not obviously;When the density set up to lOμg/ml, the apoptosis of EPCs advances manifest;When 20μg/mlβ-elemene affect EPCs 24h even 48h, the apoptosis of EPCs advances obviously too.五. The effect ofβ-elemene on the cell cycle of EPCs induced by tumor cells supernatantAffter different density(5,10,20μg/ml)β-elemene affect EPCs 48h and 20μg/ml P-elemene affect EPCs different time(12,24,48h). Compare to the control, the ratio of cells in Gl phase rises greatly (P＜0.05) but in S phase decrease obviously (P＜0.05).In a word, the EPCs cell cycle was arrested in G1 phase.六. The effect ofβ-elemene on the VEGF and VEGFR-2 mRNA expression of EPCs induced by tumor cells supernatantAffter different density(5,10,20μg/ml)β-elemene affect EPCs 48h and 20μg/ml P-elemene affect EPCs different time(12,24,48h). Compare to the control,the VEGF and VEGFR-2 mRNA expression of EPCs decreased obviously (P＜0.05)Conclusions1.β-elemene can restrain the proliferation of EPCs induced by tumor cells supernatant in the time and dose dependented manner;2.β-elemene can decrease tube number formation of EPCs induced by tumor cells supernatant in the time and dose dependented manner;3.β-elemene can promote the apoptosis and arrest cells entering S phase from G1 phase of EPCs induced by tumor cells supernatant in the time and dose dependented manner;4.β-elemene can decrease VEGF and VEGFR-2 mRNA expression of EPCs induced by tumor cells supernatant in the time and dose dependented manner.