| Part I To evaluate the transduction efficiency of human umbilical cord-derived late EPCs with nine recombinant adeno-associated virus serotypesObjectiveTo evaluate the transduction efficiency of (HUCB-late EPCs) with nine rAAVserotypes, and apply the rAAV serotype that has highest transduction efficiency as CXCR4 gene vector.Methods1. rAAV vector production system relies on the polyetherimide (PEI)-modified triple plasmid transfection method (transfection of 293 cells with pAAV-RC1-9, pHelper, and pAAV-CMV-GFP). Three days after HEK 293 cell transfection in the plate format, nuclei were harvested and disrupted, and the virus was then separated, purified, followed by SDS-PAGE and titration by quantitative PCR using primers specific for the GFP transgene.According to the experimental results, select the highest transfection efficiency of recombinant adeno-associated virus serotype as CXCR4 gene vector, to obtain rAAV-CXCR42. Human umbilical cord blood mononuclear cells (MNCs) were isolated by density-gradient centrifugation from human umbilical cord blood, and induced to endothelial progenitor cells (EPCs).After cultured for 4 weeks, late EPCs were obtained.3. Ten thousand late EPCs per well were plated in a 24-well plate with 500 ?l of media.Immediately after the plating, the cells were infected with 100,000 viral genomes per cell of rAAVl to rAAV9 vector. The experiment was performed in triplicate. The expression of green fluorescent protein was observed under a fluorescent microscope at 6h,12h,18h,24h,30h, and 36 h; three fields of view were randomly counted per well; and transduction efficiency (measured as percentage of GFP-positive cells) was analyzed by Image-Pro plus 6.0.Results1. Different serotypes rAAV (rAAV1 to rAAV9) expressing GFP was successfully constructed, The titer of purified rAAV-GFP were all exceeded 2.0 X 109vg/? 1,and the titer of purified rAAV6-CXCR4 were 2.88 X 109vg/?l..2. After cultured for 4 weeks. After cultured for 4 weeks. The late EPCs have been identified by morphology, phenotypes, and functions.3. Different rAAV serotypes showed different transduction efficiencies to late EPCs after 24 h of infection. Transduction efficiency reached a plateau at 24 h after infection, and transduction efficiency at 36 h versus 24 h (P>0.05), rAAV6 (0.5208±0.0147) versus rAAV2 (0.4506±0.011) at 24 h after infection (P<0.05), rAAV2(0.4506±0.011) versus rAAV9 (0.3632±0.0250) at 24 h after infection (P< 0.05).Part II Influence to angiogenesis of ischemic tissues after transplanted HUCB-late EPCs with CXCR4 gene over-expressedObjectives1. To evaluate the ability of capillary network formation of HUCB-late EPCs in vitro after transfected with rAAV-CXCR4 and rAAV-GFP.2. To evaluate whether HUCB-late EPCs with CXCR4 gene over-expressed facilitate angiogenesis in rat hind limb ischemic tissues.Methods1. Late EPCs were infected with rAAV6-CXCR^ rAAV6-GFP, and the expression of CXCR4 protein was detected by Western blot analysis.2. Three groups were set (rAAV6-CXCR4 infection group, rAAV6-GFP infection group and control group).To evaluate the ability of capillary network formation onMatrigel between the three groups.3. Mouse limb ischemia. After weighing,18 six-week-old male SD-mice were anesthetized with 10%chloral hydrate (3.5 ml/kg).The femoral artery andits branches were ligated.4. Rats were subjected to hind limb ischemic were divided into three groups randomly (experiment group, control group, blank group, and 6 rats per group). After 6 hours, experiment group were intramuscularly injected with 500ul EGM-2(contain 1X 106 CXCR4-gene transfected late EPCs). Control group were intramuscularly injected with 500ul EGM-2 (contain 1 X 106 non-gene transfected late EPCs).Blank group were intramuscularly injected with 500ul EGM-2. At 28 days after cell transplantation, animals were killed by taking off neck, and take ±calf muscles of ischemic hind limbs immediately.5. Neovascularization counting. Capillary network in hind limb muscles and next to muscles were detected by HE stain assay. The CD31 proteins, as specific markers of neovascularization, CD31 expressed in neovascularization endothelial cells were detected by immune histochemistry assay, and calculate the number of CD31 positiveneovascularization.Results1. Compared with infected rAAV6-GFP and non-infected group, the expression of CXCR4 protein was up-regulated after infected rAAV6-CXCR4.2. Three types of lateEPCsmade capillary formation on Matrigel successfully. The tube area of three groups is 0.531±0.023mm2?0.514±0.026mm2?0.523± 0.021mm2respectively. Thereis no significance of tube areain unit area among three types of LateEPCs (T>0.05).3. Compared with blank group, CD31 positive cells of new capillary network in control group and experimental group increased obviously(.P<0.05),Muscles(2.32 ±0.90?5.18±0.86?8.39±0.91) and next to muscles(5.32±0.93?8.12±0.95? 12.45±1.01)Conclusions1. Transduction efficiency to late EPCs of nine different serotypes rAAV obviously different, and rAAV6 was the highest, the next is rAAV2, and the last one is rAAV4.2. CXCR4 over-expression could promote neovascularization of HUCB-late EPCs in rat hind limb ischemic tissues. |
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