| Objective The aim of this study was to investigate the feasibility of noninvasive monitoring bone marrow mesenchymal stem cells(MSCs)transfected with tyrosinase(TYR)reporter gene after transplanted to the infracted myocardium by photoacoustic imaging(PAI),magnetic resonance imaging(MRI),and positron emission tomography(PET).Methods MSCs were harvested from the femur of Sprague-Dawley(SD)rats and obtained by using gradient centrifuge and adhesive culture methods.Flow cytometry was performed for CD4,CD90,CD31,and CD44 to identify MSCs.Lentivirus carrying a TYR reporter gene(LV-TYR)were constructed and transfected MSCs.The transduced MSCs(TYR-MSCs)were selected by puromycin.Normal MSCs were control group.TYR protein expression in TYR-MSCs was evaluated by Masson-Fontana silver staining,tyrosinase kinase activity assay and western blot.PAI and MRI of cells in different groups and concentration were performed in vitro.18F-5-fluoro-N-(2-(Diethylamino)ethyl)picolinamid(18F-5-FPN)was synthesized and cells uptake and binding were performed.Results LV-TYR reporter gene was successfully constructed and transfected MSCs.Stable cell line TYR-MSCs was successfully selected by puromycin.Tyrosinase expression was confirmed by Western blot,Masson Fontana stain and tyrosinase activity measurement.In vitro cell imaging studies revealed that TYR-MSCs showed obvious signals in PAI,while there was no signal in MSCs.TYR-MSCs treated with tyrosine showed stronger PAI signals than TYR-MSCs without tyrosine.T1-weighted MRI showed TYR-MSCs treated with tyrosine and FeCl3 together had strongest T1 signals.The signal of MSCs treated with FeCl3 had slight increasement.TYR-MSCs uptake of 18F-5-FPN was 7.86 ± 0.85% and reached peak at 60 min(n=3).Cells binding showed cells uptake could be blocked by19F-5-FPN.Conclusion In this study,LV-TYR was transfected into rat MSCs,and MSCs cell line stably expressing TYR was constructed.The PAI,MRI imaging and cell uptake experiments showed TYR-MSCs could be used to perform multimodal imaging,which provide a theoretical basis for the stem cell tracing in vivo.Objective To explore the feasibility of positron emission tomography(PET),magnetic resonance imaging(MRI),and photoacoustic imaging(PAI)in tracking the bone marrow mesenchymal stem cells transfected with tyrosinase reporter gene(TYR-MSCs)in acute myocardial infarction model of SD rats.Methods The left thoracotomy was performed and the left anterior descending(LAD)coronary artery was permanently ligated at its origin to establish acute myocardial infarction(AMI)models in SD rats.TYR-MSCs or MSCs were injected into the margin of the infarcted area 30 mins after ligation.Hematoxylin and eosin(HE)and2,3,5-Triphenyltetrazolium chloride(TTC)staining were performed 72 hours after injection.18F-FDG myocardial metabolic imaging was performed 0,6,13,20,and 27 days after the establishment of AMI models.PAI,MRI,and PET images were acquired 1,7,14,21,and28 days after cells injection to monitor TYR-MSC cells in vivo.Results The success rate of establishing AMI models reached 70% ? H&E staining of infarcted myocardium showed the myocardiocytes are necrotic,with surrounding edema and denaturation,distinct lymphocyte infiltration,myocardial fiber rupture,and vacuolar denaturation in AMI models while normal myocardial fibers were arranged in neat rows,with abundant capillaries.The area of myocardial infarction,expressed as a percent of the left ventricular mass,was determined using TTC staining,in which normal or viable reperfused myocardium stained red,and infarcted myocardium remained pale.The percent infarction was 38.1% ± 7.3%(n = 3)in rats that underwent the MI procedure,while normal rats showed no foci of abnormal pallor.Obvious absent uptake of 18F-FDG in the anterior wall was noted on myocardial metabolic imaging of the AMI animals.In vivo studies revealed that prominent signals were seen in the injected area of the myocardium on PAI/MRI/PET images,while no signal could be seen in rats injected with MSCs.Signals by these three imaging modalities lasted for 28 days.Uptake values of 18F-5-FPN in vivo only showed slight decrease from 1.78 ± 0.22 %ID/g on day1 to 1.62 ± 0.13 %ID/g on day 28 and had no statistical difference(P>0.05),while MRI and PAI signal intensity decreased dramaticly with statistical difference.From day 1 to day 28,the contrast ratio of infarcted myocardium to normal myocardium on T2*WI sequence increased from 59.18 ± 4.9 to 80.5± 8.8(P<0.05),indicating the decrease of MRI signals in infarcted area.The PAI signals decreased from 472.6 ± 48.1 on day 1 to 283.4 ± 49.2 on day 28(P<0.05).Conclusion TYR reporter gene could be used to tracking stem cells with multimodal imaging in vivo.The study showed the transplanted MSCs in infarcted area had obvious PET,MRI and PAI signals.The MRI and PAI signals decreased with time while PET signals maintained stable. |
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