| BackgroudEndothelial progenitor cells(EPCs) and hemopoietic stem cells(HSC) come from the same ancestor—angioblast which exists in the mesoblast. EPCs are mainly located in bone marrow postnatal. Bone marrow-derived EPCs were firstly identified from peripheral blood using magnetic micro beads coated with CD34+ antibody by Asahara in 1997. EPCs can be isolated from bone marrow, umbilical cord blood and peripheral blood using the magnetic micro beads, adherence culture and flow cytometry.We always use adherence culture to culture EPCs and get worderful effect. Gliomas are the most common malignancies of the central nervous system in humans. Gliomas is characterized as a highly angiogenic tumor; and this extensive blood vessel growth is critical for tumor progression and invasion. Growing body of evidence indicates that neovascularization processes associated with tumor growth are in part supported by recruitment of bone marrow-derived, endogenous endothelial progenitor cells(EPCs) and their functional incorporation into the new vasculatures. Due to their ability to self-renew, circulate, home to the tumor sites, and differentiate into mature endothelial cells(ECs), EPCs hold enormous potential to be used as a diagnostic and/or therapeutic agent in anti-tumor therapies. Hence, this review focuses on EPCs and their role in tumor angiogenesis with the emphasis on EPC recruitment/migration, the potential use of EPCs as a therapeutic tool and imaging probe. The aim of this study was investigating the dynamic homing characteristic of exogenous endothelial progenitor cells(EPCs) in rat glioma in vivo to provide an experimental basis for the feasibility of using magnetically labeled EPCs as MRI target tracing vectors.Purpose : The aim of this study was to investigate the dynamic homing characteristic of exogenous endothelial progenitor cells(EPCs) in rat glioma in vivo, and whether the migration of EPCs is related to the expression of angiogenic and other tumor or surrounding tissue microenvironmental factors. The particular advantage when creating EPC based therapeutic approach comes with the prospect that these cells can be used as therapy target, gene/protein delivery vehicle and imaging probe.Materials and methods:1 、 Dynamic MRI research of EPCs tracing rat glioma and the homing of Magnetically Labeled Endothelial Progenitor Cells in Glioma.1.1 Isolation, Culture, and Identi?cation of Rat Spleen-originated EPCUsing the methond of our Research Group Dr Fang.1.2 Establishment of Glioma Model48 male Sprague-Dawley ratsweighing 140±20 g were randomized into the fake group,experimental group and control group. For experimental and control groups, amicrosyringe of C6 cells was inoculated in the right caudate nucleus through a 1-mm burr hole at 1 mm in front of the bregma and 3 mm right of the midline, and of fake group,only DPBS was injected in the brain. 2×106 USPIO-EPCs was transplanted via the tail vein to experimental and control groups.1.3 MRI Scan finding for Rats with Glioma7.0T MRI system(Biospec70/20USR) were used in our research, and T1 WI,T2WI,T2 map and SWI sequences were involved. Both groups were scanned on days 1, 3, 5, 7 after the transplantation of USPIO- EPCs, the variation of the singal was noticed in every sequence.Measure the T2 value in T2 map sequence and draw the time-T2 value curve.1.4 Pathologic Analysis of the gliomaThe rats sacri?ced at each time point and perfused transcardially with paraformaldehyde in phosphate-buffered saline. The samples were treated with immunohistochemical staining for studying vascular endothelial growth factor(VEGF),MMP-9 and SDF-1 expression.All groups were treated with Prussian blue staining at the 4 time point. all the Prussian blue staining positive samples were accepted F4/80 stain to distinguish USPIO-EPCs from macrophage which swallow the iron.We notice the distribution of USPIO-EPCs and VEGF, MMP-9 and SDF-1 positive cells and evalute the their relationship.2、The Effects of USPIO-EPCs on Growth of Glioma in high field intensity MRI2.1 Establishment of Glioma ModelAccording to the Part 1, 8 days after the glioma models were established, in the group A, 2×106 and 3×106 USPIO-labeled EPCs were transplanted via the tail vein in group A and Group B, equal dose of DPBS was transplanted via the tail vein in control group C.2.2 The variation of the tumor volume in the 4 time pointEnhanced T1-weighted imaging was performed using the same parameters to measure the tumor left-right diameter,front-back diameter, vertical diameter,and get the glioma volume.2.3 DSC-EPI image analysisThe dynamic suscepti-bility contrast(DSC) perfusion data were managed for calculation to formulate the pseudocolor images of cerebral blood volume(CBV) andmean transit time(MTT).The CBVimages were contrasted with the enhanced images of the same rat to obtain 5 areas of interest areas. Mean values were calculated to obtain the CBV and MTT values of the same tumor. The CBV values of the two sides were compared to obtain the relative CBV(rCBV) of the tumor.2.4 Pathologic Analysis of gliomaThe samples were treated according to Part1,the microvascular density(MVD) and diameter of microvessels were determined using Image-Pro Plus version 6.0,the densest area of neovasculature was selected under low-power light microscopy,and the numberofmicrovessels staining in brownin the hot spots was counted under high-power light microscopy(200×). The mean count of microvessels in three to ?ve ?elds was the MVD. Inaddition, ?ve high-power?elds(200×)in the hot spots were randomly selected to measure the microvascular diameter and determine the mean value.Results1、dynamic homing characteristic of USPIO-EPCs in rat glioma1.1 The MRI scanning in dynamic homing of USPIO-EPCs in rat gliomaIn the fake experimental group, the MRI signal was similar before and after EPCs admistration, The T2 value-time curve was straight. in the experimental group, hypointense areas were detected at the periphery of the tumor on the1 day after transplantation ofUSPIO- EPCs, and more hypointense areas were observed in the center of the tumor over time. The T2 value-time curve was downtrending.The volume of the tumor increase without singal change in the control group gradually.There were a little blue-stained cells in fake experimental group, and several bluestained cells were observed at the the periphery of the tumor on the ?rst day after transplantation of EPCs, and migrated in the center of the tumor gradually. There was signi?cant difference in the number of blue-stained positive cells among the two groups(P<0.01).based on the result of F4/80 staning, the blue stained is USPIO-EPCs not the macrophagocyte.1.3 Immunohistochemical Analysis of the USPIO-EPCs tracing rat gliomaBoth SDF-1 and MMP-9 showed generalized expression in the periphery of the tumor in the early stage, and gradually increased in the center of the tumor over time. Compared to VEGF, VEGF positive cells gradually increased over time,but we don’t found the distribution transformation.2. The effects of magnetically labeled endothelial progenitor cells on growth of glioma in dynamic MRI2.1 The effecet of different dose of USPIO-EPCs on glioma gowingThere was no signi?cant difference in the volume of the glioma among the three groups(P >0.05)in 1,3,5,7 days after USPIO-EPCs transplantation.2.2 The effecet of different dose of USPIO-EPCs on PWIThe rCBV value of the tumor parenchyma increased over time,and there was signi?cant difference in comparisons at four different time points(P < 0.01). However, there were no remarkable differences in rCBV values between 3 groups(P >0.05).2.3 The effecet of different dose of USPIO-EPCs on glioma pathological feature in the different phaseYellow or brown particles in the cytoplasm were considered to represent positive cells for VEGF and MMP-9 staining. The number of positive cells was counted under high-power light microscopy(200×) using Image-Pro Plus 6.0. The mean value was determined to be the number of cells positive for VEGF and MMP-9 expression at four point time. MVD in each group was signi?cant different at different time points(P <0.01), but MVD was not markedly different among the three groups at the same time point(P >0.05).Microvascular diameterwas determined on day 14 after transplantation of tumor samples in the hot spots in all four groups and ranged from 17.25—24.38μm. There was no remarkable difference in microvascular diameter among the there groups(P >0.05). 1.2 Pathologic Analysis of the USPIO-EPCs tracing rat gliomaConclusion1、USPIO-EPCs can home to the the area of giloma, the process can be detected by MRI. Hypointense areas were detected at the periphery of the tumor on the1 day after transplantation ofUSPIO- EPCs, and more hypointense areas were observed in the center of the tumor over time. The distribution area of MMP-9 and SDF-1 positive cells are similar to the distribution of prussian blue positive staned cells,so it indicated SDF-1 and VEGF have been shown to be a potent chemoattractant for USPIO-EPCs.2、The method of building glioma involved brain injury,which is the reason of EPCs homing.wo count the number of EPCs in the experiment group and the fake group, we found remarkable difference in number among the two groups(P <0.01),it indicated the reason of EPCs recruitment is the glioma not the injury. F4/80-positive cell were not positive for iron by Prussian blue staining. These ?ndings indicate that the Prussian blue positive cells that migrated to the tumor periphery were viable cells and did not release iron oxide nanoparticles that could be phagocytosed by the macrophages.3、Both magnetic resonance imaging and immunohistochemical ?ndings con?rmed different-dose of EPCs could not affect thebiologic behavior of C6 glioma cells including r CBV,MVD, microvascular diameter,the variation of VEGF and MMP-9.The reason is complicated,involving the microenvironment in vivo and the diverse mode of vascularization. Therefore, exogenous EPCs could not exert signi?cant promoting effects on glioma growth,it is safety to use the certain EPCsas the therapeutic gene carrier in the early glioma. |
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