| Objective:(1) To investigate the regulation of Dll4 and Notchl mediated by Hepatocyte growth factor(HGF) in human femoral artery endothelial cells(HFAECs),and to establish the basis for further investigating the mechanism of HGF promoting arteriogenesis;(2) To investigate the relationship between the proliferation and migration ability of artery endothelial cells and Dll4-Notch-Hey2 signaling pathway in the process of arteriogenesis,and elucidate the mechanism of HGF promoting arteriogenesis.(3) Endothelial progenitor cells(EPCs) were isolated,cultured and identified in vitro,and which were used as a study model from the point of angiogenesis,to further investigate that HGF promoted the formation of artery whether via Notch signal pathway regulating the differentiation of progenitor cells or not.Methods:Part 1 HFAECs were cultured in vitro and infected with the different multiplicity of infection(MOI) of Ad-GFP(50,100,150,200,250,300 pfu/cell).The optimal MOI was screened and determined by MTT(detecting the degree of cell damage) and flow cytometry(detecting the rate of transfection),and used as the optimal MOI of Ad-HGF.At 48 h after HFAECs were infected with the optimal MOI of Ad-HGF,HGF expression level in the supernatant was detected by ELISA;meanwhile,the transcription of Notchl and Dll4 genes was detected by reverse transcription-PCR(RT-PCR).Ad-GFP-infected HFAECs were used as negative controls. Part 2 On account of prophase study,cells and supernatant were harvested at the indicated times(0,24,48,72 h) after HFAECs were infected with the 200 pfu/cell of Ad-HGF.HGF expression in the supernatant was detected by ELISA and the transcription of Notchl,Dll4 and Hey2 genes was analyzed by RT-PCR.The changes in the proliferation,survivorship and migration ability of HFAECs,and the relationship between Dll4-Notch-Hey2 signaling pathway and them was detected respectively by MTT and Transwell migration experiment.Ad-GFP-infected HFAECs were used as negative controls.Part 3 Cord blood,asepstic,anticoagulated by Heparin,was used as the example resource of EPCs.After diluting with PBS(1:1),7 mL of cord blood was added slowly into centrifuge tube which containing 3mL of lymphocyte isolation.To centrifuge for 30 min at 2000 r/min and collect the mononuclearcells in cloudiness buffy coat.Cells were inoculated in 6-well plate coated with human plasma fibronectin purified protein and cultured in M-199 medium.To observe the morphologic change of adherent cells and identify EPCs by immunocytochemical method.Experiment results were presented as mean±SD.Statistical analysis was performed using the SPSS 13.0 software package.Results:(1) The optimal MOI was 200 pfu/cell according to the high rate of transfection and the low degree of cell damage;the ELISA results showed that,Ad-GFP group: (3.672±0.810) ng/ml;Ad-HGF group:(86.318±3.864) ng/ml.At 48h after transfection,HGF expression level had statistical difference between two groups(P＜0.05);(2) After Ad-HGF transfected HFAECs for 48h,the RT-PCR results showed that the transcription level of Notchl and Dll4 genes was obvious up-regulation in Ad-HGF group;the results of gel-image analytical system showed that Dll4: 0.788±0.021 and 0.936±0.018;Notchl:0 and 0.458±0.023 respectively in Ad-GFP and Ad-HGF groups,and the transcription level of objective genes had statistical significance(P＜0.05) by half t-test was performed between two groups.(3) After Ad-HGF transfected HFAECs at the indicated times,the ELISA results showed that Ad-HGF effectually transduced into HFAECs,and HGF expression level had time-dependence within 48 h,and began to decrease at 72 h;(4) After Ad-HGF transfected HFAECs at the indicated times,the RT-PCR results showed that the transcription of only Dll4 gene was detected at 0 h;the transcription of Notchl and Dll4 genes had time dependence within 48 h;the transcription of Notchl gene had not been detected and Dll4 weakened obviously at 72 h;kinetics of Hey2 induction by HGF was consistent with that of Notchl.The results of gel-image analytical system were consistent with above-mentioned observation results;(5) In MTT experiment of Ad-HGF-infected HFAECs at the indicated times,it was initially observed comparing with the Ad-GFP group that the proliferation and survivorship ability of HFAECs enhanced obviously.The transwell migration experimental results showed that the migration ability of HFAECs between two groups was not significantly different in the absence of HGF at the indicated time (p＞0.05);but the migration ability of HFAECs in Ad-HGF group was obviously more enhanced than that in the control group in the presence of HGF at 24 h and 48 h(p＜0.05),and that at 48 h was more enhanced than at 24 h in Ad-HGF group but had no,significant difference(p＞0.05).(6) The results of observing the morphous of EPCs cultured in vitro showed that cells were fusiform shape and the appearance of "stones paved roads" with the lasting of cultivation time,as was consistent with the report results of records.(7) The result of identifying EPCs by immunocytochemical method showed that VEGFR-2,CD34 and CD133 of isolated cultured cells were all positive.Conclusion:(1)HGF had the up-regulation effect on the transcriptional level of Notchl and Dll4 genes in HFAECs:suggesting that the mechanism of HGF promoting arteriogenesis might be related with Notch signaling pathway.(2) By activating Dll4-Notch-Hey2 signaling pathway,HGF indirectly prolonged cell survival,promoted the proliferation and migration ability of cells to promote the formation of offspring artery branching.(3) Cells,which were isolated from cord blood by density gradient centrifugation and cultured,had the character of EPCs,and might be used as the further study model in vitro. |
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