Rehabilitation Of Injured Endothelium Through Transplantation Of Endothelial Progenitor Cells Labeled By Superparamagnetic Iron Oxide Nanoparticles

Injured endothelium is the triggering factor of cardiovascular diseases such as atherosclerosis, restenosis et al. Pathological characteristic is that matrix underneath intima exposes to blood due to damaged endothelial cells, leading to accumulation of platelet releasing mitogen by degranulation, which vice versa induces aggregation of platelet and granulocytes and stimulates proliferation, migration and secretion of smooth muscle cells and remodeling of vessel wall. At the same time, the integrity destroy of endothelium layer causes to decreasing of auto-secreted protective transmitter (NO, PGI, et al) while increasing of contractive transmitter and growth-promotion substances. Studies elucidated that the rapidity of early lumen re-epithelialization after injury plays a key role in the local intimal hyperplasia. How to accelerate the damaged lumen epithelialization in order to rehabilitate endothelial cells and inhibit smooth muscle cells is interesting to vascular scholars.Endothelial progenitor cells originate from bone marrow, releasing into circulation after mobilization, homing to injured site and differentiating. Lots of data suggest that EPCs plays an important role in angiogenesis and intimal repair. How to harvest enough EPCs and ensure their viability and biological characteristic stable meanwhile avoid abnormal differentiation in vitro is still a tough problem. In addition, tracking of EPCs can evaluate biological distribution and proliferation and provide feedback into the preferred sites of engraftment, which will aid in determine the optimal dosing schedule and route of transplanting cells so that achieve the desired therapeutic outcome.Magnetic resonance imaging (MRI) is the most commonly used imaging modality for in vivo tracking of labeling stem cells, because it is noninvasive, generates high-resolution images, and does not rely on radioactive isotopes, which may be an important advantage for longitudinal studies. Our studies focus on biological distribution of transplanting cells in vivo after labeling cells with magnetic particles in vitro. Firstly, we isolate mesenchymal stem cells and induce them into EPCs with optimal stimulation. After that we administrate the EPCs labeled with superparamagnetic iron oxide(SPIO) nanoparticles to the injured carotid artery, tracking these labeled cells and evaluating their effect of endothelial repair.PartⅠIsolation, Cultivation and Identification of Endothelial Progenitor Cells Objective:To study in vitro the isolation and cultivation of mesenchymal stem cells(MSCs) from rabbit bone marrow and differentiation with inducible factors, identifying and expanding numbers of endothelial progenitor cell(EPCs). Methods: MSCs were extracted from bone marrow by density gradient centrifugation and the method of adherence-sieve. Differentiation into endothelial cell colony unit was controlled by the committed induction with VEGF and bFGF. Analyses with flow cytometry, immunofluresence and immunohisrochemistry assay were carried out after seven-day culture. Results:Adherent cells were round after 24 hours and cell colony were formed by spindle-like cells radiating towards periphery in 72 hours; whirlpool or trabs-like cell colonies were found in 5th day and endothelioid cell colonies link with each other, even showing a tube-like structure in 7th day. CD34 and CD 133 were positive in flow cytometry with 2.16±0.09%; the expression rate of KDR of differentiated cells was 72.56±8.76%, while the spindle-like cells were reactive to Dil-ac-LDL and FITC-UEA-1. Conclusion:Bone marrow-derived MSCs, isolated and cultivated with the methods of density gradient centrifugation combined adherence-sieve, can differentiate into EPCs with inducible factors in vitro.Part IIBiological Experiments of EPCs Labeled by SPIO Nanoparticles and NC-GFP-Lentivirus in vitroObjective:To evaluate the effect of toxicity and viability of EPCs labeled with superparamagnetic iron oxide (SPIO) nanoparticles and NC-GFP-Lentivirus. Methods: EPCs labeled with different SPIO concentration and multiplicity of infection (MOI) of NC-GFP-Lentivirus were evaluated by MRI signal intensity, viability test, proliferation assay, LDH release experiment, migration assay, cell cycle and apoptosis analysis. Results:the MRI signal intensity in T2WI decreased with the increasing SPIO concentration and it is difficult to discriminate over 50ug/ml and 100ug/ml. The viability also reduced while the release of LDH increased, and the proliferation, migration, cell cycle and apoptosis were not affected even though with the higher SPIO concentration. The viability is significantly different (P<0.05) with MOI=50, compared to the other groups, and the release of LDH is 10.34% at MOI=50, which indicates the toxicity to cells. The difference of apoptosis is significant (P<0.05) at MOI=2 and MOI=50, while migration also reduced greatly. The proliferation is not affected under different MOI. Conclusion:EPCs can be labeled with SPIO nanoparticles (Fe2+=25ug/ml) and NC-GFP-Lentivirus(MOI=10).PartⅢRehabilitation of Injured Endothelium through Transplantation of Endothelial Progenitor Cells Labeled by Superparamagnetic Iron Oxide Nanoparticles in MRI Objective:To survey the effects of the endothelial progenitor cells (EPCs) for repairing arterial intimal injury. Methods:EPCs, labeled by the superparamagnetic iron oxide (SPIO) and green fluorescent protein (GFP), were injected into the injury artery lumen. At Id,7d and 28d after cell transplantation, magnetic resonance imaging (MRI), H&E, Prussian blue staining, biopsy, immunohistochemistry, immunofluorescence and western blot for endothelial nitric oxide synthase (eNOS) were applied to evaluate the intimal restoration. Results:The low signal intensity was observed in the transplantation position in T2WI. EPCs in transplantation group adhered to injury lesion and differentiated into endothelial cells as time passed, which was verified by H&E, Prussian blue staining, vWF immunohistochemistry, GFP immunofluorescence, meanwhile expression of eNOS of the transplantation group increased with time course. Conclusion:MRI could be used to track the EPCs labeled by SPIO with non-quantitative analysis; the restoration of the arterial intima could be accelerated by bone marrow derived EPCs transplantation.