| Part1 Construction of recombinant adeno-associated viruses vector carrying CXCR4 geneObjective:1.CXCR4 gene was cloned into the recombinant adeno-associated virus(AAV)vector as a therapeutic tool to enhance the migrational potentiality of EPCs.2.Polyethyleneimine(PEI)-mediated triple plasmids packaging system was transfected into human embryonic kidney 293 cells(HEK293 cells)to explore the optimal transfection conditions.Methods:1.CXCR4 gene was amplified by RT-PCR and cloned into the plasmid pAAV2.1-CMV-LacZnls to construct plasmid pAAV2.1CMV-CXCR4 before identified by the assay of double digestion,PCR and sequencing.2.Meanwhile,we transfered packaging system comprised of pAAV2.1CMV-GFP,pAAV-RC2 and pHelper into HEK293 cells mediated by PEI to detect the transfection efficiency of different total plasmid DNA dosages(1,2,3,4,5,6 ? g)and different PEI/Plasmid ratios(1:1,3:1,5:1,7:1).ImagePro Plus6.0 Software were used to calculate the transfection efficiency by detection of green fluorescence protein(GFP).Transfection efficiency of the optimized transfection system was further observed in different time points(12,24,36,48,60,72h).3.Then,rAAV2-CXCR4 and rAAV2-GFP was packaged in the optimal transfection conditions and purified by PEG8000/NaCl/chloroform assay before identified by SDS-PAGE and titered by qPCR.Results:1.The recombinant plasmid pAAV2.1-CMV-CXCR4 was successfully constructed and identified.2.Total plasmid dosage of ?g/well with PEI/Plasmid ratio of 3:1-5:1 was an efficient transfection condition.Transfection efficiency-Time curve was an S-shaped curve.Transfection efficiency reached plateau at 60h after transfection.3.The concentration of purified rAAV2-CXCR4 and rAAV2-GFP was 2.8184×109vg/?1 and 3.2211×109vg/?1,respectively.Part2 Isolation,culture and identification of endothelial progenitor cells from umbilical cord bloodObjective:1.Mononuclear cells(MNCs)were seperated from umbilical cord blood and induced to differentiate into endothelial progenitor cells(EPCs).Methods:1.After being isolated from cord blood by density gradient centrifugation with lymphocyte separation liquid,the MNCs were planted on fibronectin-coated six-well plates and induced to differentiate into EPCs in vitro with endothelial cell growth medium.Four days after primary culture,the nonadherent cells were discarded with the change of medium.Then the medium was changed twice to thrice a week.2.Morphological characteristics were observed under phase-contrast microscope.3.Expression of cell markers,including CD34,KDR and CD14 were detected by flow cytometry assay.4.The ability to uptake Dil-acLDL and to bind FITC-UEA-1 were further identified by the laser scanning confocal microscope system.Results:1.The mononuclear cells plated on fibronectin promptly attached and became spindle-shaped within four days accompanied by the appearance of blood island-like cell clusters comprised round cells centrally and sprouts of spindle-shaped cells at the periphery.The formation of cellular networks and tube-like structures appeared after two weeks of culture.When cultured for up to three weeks,the proliferated cells showed cobblestone like appearance and exhibited colony formation ability after passage.2.Flow cytometric analysis revealed that the induced cells were positive for CD34 and KDR,while negtive for CD 14.3.Dil-Ac-LDL-incorporated(red)and FITC-UEA-1-binding(green)cells were observed under confocal microscope.Part3 Infection of EPCs with recombinant adeno-associated virus carrying CXCR4 gene in vitroObjective:1.Stromal cell-derived factor-1(SDF-1)and its receptor CXCR4 were considered as critical mediators for the recruitment of circulating progenitor cells,which direct the study to investigated the effect of CXCR4 over-expression on SDF-1-induced EPCs migration.2.rAAV2-GFP was transfered into EPCs to explore the optimal transfection conditions.Methods:1.ImagePro Plus6.0 Software were used to evaluate the infection efficiency by detection of GFP signal after transfer of rAAV2-GFP of different MOI(10,000vg/cell and 100,000vg/cell)into EPCs.2.Then,EPCs were infected with rAAV2-CXCR4 at the optimal MOI before detecting CXCR4 protein by Western Blot assay.3.After that,EPCs were divided into three group,namely,rAAV2-CXCR4 infection group,rAAV2-GFP infection group and Control group.The effect of CXCR4 over-expression on EPCs'proliferation capacity were detected by MTT method and its effect on EPCs' migration were determine by transwell assay.Results:1.The proportion of GFP-visible EPCs in the low MOI(10,000vg/cell)group and high MOI(100,000vg/cell)group is 0.2064±0.0266 and 0.3876±0.0242,respectively,at 12 hours after infection;and increased to 0.3459±0.0160 and 0.5941±0.0175,respectively,at 24 hours after infection.No further increase was observed when prolonged infection time.Infection efficiency of the high MOI group was higher than that of the low group(P<0.05)at each time points.2.EPCs in rAAV2-CXCR4 infection group showed a higher CXCR4 expression when compaired to both the rAAVGFP infection group and PBS group.3.EPCs proliferation curve in the control group was an S-shaped curve;infection efficiency reached plateau at 72h after infection.Compared with the control group,EPCs proliferation in both rAAV2-CXCR4 infection group and rAAV2-GFP infection group was significantly impaired with the time to reach plateau significantly delayed.4.Both low concentration(10 ng/mL)and high concentration(100 ng/mL)SDF-1a-induced EPCs migration in vitro was dramatically enhanced by rAAV2-CXCR4 infection as opposed to the control group(416 ± 33 V.S 234 ± 16,P<0.001;504±21 V.S 413 ± 15,P<0.001),with more EPCs attracted in low concentration group than that in high concentration group(181±25 V.S 91±34,P<0.001).Whereas rAAV2GFP infection had no significant effect on SDF-la-induced EPC migration in vitro compared with the control group(218±18 V.S 234±16,P>0.05;384 ±16 V.S 413 ± 15,P>0.05).In the absence of SDF-la,there was no significant difference between each groups in baseline EPCs migration quantity(53±14 V.S 55 ± 19 V.S 51±25,P>0.05).Conclusion:1.CXCR4 overexpression enhances SDF-1-induced EPCs' migration.2.CXCR4 overexpression has no effect on EPC's proliferation in vitro. |
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