| BackgroundCell transplantion is a hot spot in many fields.With the development of conceptions and technique over 30 years,stem cell transplantion had been applided to clinical setting from basic research.Since endothelial progenitor cells(EPCs),which can differentiate to endothelial cells, were first separated and cultured from peripheral blood in 1997,many researchs have been shown that EPCs can be used in vascular tissue engineering,coronary heart disease,tumour,trauma healing and so on. In view of the value of EPCs for clinical appilication , EPCs trasplantion has a glorious prospect in the treatment of some diseases to improve the quality of life or life time .However, in vivo image of transplant cells and future investigation would be a trouble to be faced with. In this study,we have performed EPCs labeled with ultrasuperparamagnetic iron oxide/ Ferumoxides,P7228 and paramagnetic Gd chelate(Gd-BOPTA) in vitro MR imaing,to investigate the method of labeling EPCs and in vitro MR imaging, and provide experimental evidences for EPCs transplantion in the clinical application.ObjectiveTo investigate the method of the isolation,cultivation,identification of EPCs derived from hunman umbilical cord blood and the best concentration for the growth of EPCs by comparing the different inoculated concentration,and provide the platform of the experimental study and the clinical practice;To investigate the labeling methods of Ferumoxides,P7228,Gd-BOPTA and the influence of different concentration or different incubation time of labeling EPCs on viability,adherence,immigration,proliferation of cells,and explore the optimal labeling concentration and the best labeling time;To investigate imaging characterization of labled cells by the technique of magnetic resonance imaging in vitro, and identify the optimal MR contrast agent and the best scanning sequence;To provide the theory and experimental evidences for EPCs transplantion in the clinical application.Materials and methods1.Isolation,cultivation,identification and cytobiological characteristics of EPCs derived from human umbilical cord bloodHuman umbilical cord blood from healthy parturients(be approved)eutocia or abdominal delivery were diluted with DPBS in 1:1, mononuclear cells were isolated by density gradient centrifugation, inoculated on culture plate coated with human fibronectin by different concentration, cultivated in endothelial growth medium-2 supplied with recombined human VEGF,IGF,b-FGF,FBS, to observe the information of cell adherence and colony;After 3 days cultured,non-attached cells were washed by PBS and then attached cells were cultured in EBM-2MV for 7 days,the medium was changed every 3 days,the cell's morphology were observed by microscopy.EPCs were characterized as adherent cells double positive for DiI-acLDL uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope.EPCs were documented by demonstraining the expression of cell surface makers with flow cytometry.EPCs migration was assayed with modified Boyden Chamber assay. EPCs adhesion assay was performed by replating those on human fibronectin-coated culture plate, and then adherent cells were counted.2.The study on cytobiological characteristics of EPCs labeled with Ferumoxides,P7228 and Gd-BOPTAEPCs were labeled with Ferumoxides,P7228 and Gd-BOPTA, ploy-L-lysine(PLL),jetPEITM-FluoF as transfection agents, respectively.The ratio of contrasts to transfection agents was 1:0.03.The mix were dulited by medium ,and then the finial concentrations of contrasts,transfection agents would be 5μg/ml,12.5μg/ml,25μg/ml,32.5μg/ml,50μg/ml and 0.15μg/ml,0.375μg/ml,0.75μg/ml,1.125μg/ml,1.5μg/ml,the labeling time was 24h,48h,72h, respectively.Idenfication of Fe or Gd particles in cells and the labeled efficiency of different labeling concentrations or different labeling time were assayed with Prussian blue staining,fluorescence microscope and transmission electron microscopy.The viability of labeled cells were documented by Typan blue dye exclusion test.Labeled EPCs proliferation,migration were assayed with 3-(4,5-dimethtiazol-2-yc)-2, 5diphenyltetrazolium bromide(MTT)assay,modified Boyden Chamber assay respectively. Labeled EPCs adhesion assay was performed by replating those on human fibronectin- coated culture plate,and then adherent cells were counted.3. The study on MR imaging of EPCs labeled with Ferumoxides,P7228 andGd- BOPTA in virtoSamples of un-labeled cells and labeled EPCs with different concentration of Ferumoxides,P7228,Gd-BOPTA after labeled 24h were imaged by MRI with T1WI,T2WI and T2*WI sequence , the signal intensity were imaged and statistically analyzed. Results1.Isolation,cultivation,identification and cytobiological characteristics of EPCs derived from human umbilical cord bloodThe lately-isolated MNCs were round, and parts of the cells started attaching after 24h.After 3 days cultured, attached cells become bigger and more lucency , some were fusiform. After 7 days cultured, the attached cells were able to form cell colonies and present spindle-shaped.After 14 days cultured, the cells got close to coalesce and exhibited the typical cobblestone morphology.The best inoculated concentration for cell adherence and cell colony was 2.5×106/ml. EPCs were characterized as adherent cell double positive for DiI-acLDL uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope, the proportion of cell double positive was 95%. The expression of CD34,,VEGFR2(KDR),AC133 with flow cytometry were (1.67±0.35%),(12.63±9.70%),(66.81±6.63%)。The results of EPCs migration,adhesion were (33.7±1.6),(29.2±1.2).2.The study on cytobiological characteristics of EPCs labeled with Ferumoxides,P7228 and Gd-BOPTAThe EPCs could be labeled with Ferumoxides,P7228,Prussian blue staining showed numerous blue-stained iron particles in the cytoplasm. The labeling efficiency become higher with the increasing of labeling concentration and the labeling time elapsed, and the efficiency of P7228 was higher than FE. At the concentration of 25μg/ml after labeled 12h,24h, the labeling efficiency of FE,P7228 were (91.3±0.59%),(93.2±0.37%) and (96.7±1.21%),(97.0±1.1%),respectively. With the increasing of labeling concentration and the labeling time elapsed ,electron microscopy observation showed that the vesicle-like structure with membrance involving the Ferumoxides,P7228 in the cytoplasm became more and more, which mean that the iron content also increased.The EPCs could be labeled with Gd-BOPTA and fluorescence microscope observation showed that green fluorescence particles could be seen in the cytoplasm, electron microscopy observation showed numorous Gd particles were located around Golgi apparatus or endosome of cell membrane.The labeling efficiency was lower than SPIO.The labeling pattern of Gd-BOPTA was parabolic, the labeling efficiency became higher with the increasing of labeling concentration and the labeling time elapsed,it achieved the peak at the concentration of 25μg/ml labeled 24h,then it became lower. At the concentration of 12.5μg/ml,25μg/ml,37.5μg/ml after labeled 12h,24h ,the labeling efficiency of Gd-BOPTA were (55.7±0.79%),(80.8±0.51%),(71.6±0.64%)and(67.4±1.67%),(88.2±1.56%),(73.2±2.17%),respectively.They were lower than FE and P7228 at the same condition.At the concentration of 25μg/ml,37.5μg/ml and 25μg/ml after labeled 24h,the viability of cells labeled with FE,P7228,Gd-BOPTA is 92.4%,89.5% and 93.7% respectively.EPCs proliferation,migration and adhesion were not influenced by FE,P7228,Gd-BOPTA under the concentration of 25μg/ml,37.5μg/ml,25μg/ml(P<0.05)3.The study on MR imaging of EPCs labeled with Ferumoxides,P7228 and Gd-BOPTA in vitroThe SI of cells labeled with FE,P7228 on T1WI became incresed lenitively,there was no significant difference (P>0.05);The SI of cells labeled with Gd-BOPTA became increased first and then decreased following the increasing concentration,the SI at the concentration of 25μg/ml(161%)was maximal,and there was a significant difference with unlabeled cells(P<0.05).The SI of cells labeled with FE,P7228 on T2WI and T2*WI became qucikly decreased following the increasing concentration, there was nearly no SI at the concentration of 50μg/ml labeled with P7228 on T2*WI.At the concentration of 5μg/ml,the△SI of FE,P7228 were -10.4%,-17.8% and -12.3%,-34.9%,respectively.The△SI on T2*WI was more sensitive than on T2WI, P7228 better than FE .The SI on T2*WI became decreased following the increasing concentration of FE-PLL,P7228-PLL(r= -0.681,-0.712,P<0.05).There was no significant difference on△SI of Gd-BOPTA between T1WI and T2WI,T2*WI(P>0.05)。 Conclusions1.Combined Ficoll'density gradient centrifugation with adherence screen,we could get optimal purity and survival rate of human umbilical cord blood derived EPCs.We could get optimal differentiation and proliferation with 2.5×106/ml inoculation concentration.2.EPCs can be labeled efficiently with FE,P7228 and Gd-BOPTA mediated by PLL,jetPEITM-FluoF,respectively.No influence on biological activity under suitable concentration. The optimal labeling concentration of FE,P7228 and Gd-BOPTA are 25μg/ml,37.5μg/ml,25μg/ml,the best labeling time is 24h.3.The major influence of superparamagnetic iron oxide on MR singal intensity is T2WI and T2*WI effect, the sequences of T2WI and T2*WI are specific for revealing of ultrasuperparamagnetic iron oxide agent,also representing the quantity roughly.The negative effect becomes significant following the increasing concentration of SPIO ,P7228 is better.The sequence of T2*WI is the optimal MR sequence for ultrasuperparamagnetic iron oxide agent. The major influence of paramagnetic Gd chelate (Gd-BOPTA) is T1WI, the postive effect becomes significant following the increasing concentration of Gd-BOPTA ,but the effect becomes decreasing after the maximal SI.So there is the optimal labeling concentration of Gd-BOPTA for the best MR image.4.The labeling method of SPIO is more simple and effective compared to the method of paramagnetic Gd chelate;the labeling efficency of P7228 is better than FE,the influence on labeling cell is lower,and the variance of SI on MR imaging is more sensitive.So P7228 is more suitable for cell labeling. |
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