| Background and objective:Malignant glioma is the most common primary brain tumor with strongest invasion in adults. Tthe invasive growth pattern of malignant glioma is one of main factors for post-operative recurrence, poor results of radiotherapy and chemotherapy and high mortality in patients. It is important to improve the accuracy of early diagnosis and find new treatment strategies towards glioma. Recent research results showed that the angiogenesis is key to growth, invasion and metastasis of the tumor. Molecular targeted anti-angiogenesis to glioma becomes a hot research spots and search for targeted delivery of vector is undoubtedly of high application value. Endothelial progenitor cell (EPCs) is a precursor cell that has a highly proliferative potential. EPCs can differentiate into vascular endothelial cells under condition culture. During development of the glioma, EPCs can be integrated/homed into tumor vascular zone to take part in angiogenesis. Therefore, EPCs may become an important imaging diagnostic marker as well as a new carrier of anti-tumor therapy.In our study, we firstly set up an in vitro culture system of umbilical cord blood EPCs. Then EPCs were labeled with superparamagnetic iron oxide (SPIO) mediated by Poly-L-Lysine (PLL). After the cells was grafted into nude mice glioma model via the end of vein, 1.5T MRI was used to track the labeled cells in vivo to explore the ability of EPCs in chemotactic migration to glioma, analyze the temporal-spatial relationship between EPCs and glioma angiogenesis, and track the distribution and outcome of EPCs in tumor-bearing nude mice, which may provide the experimental and theoretical basis for the application of EPCs as a vector of target therapy of the glioma.Methods: (1)EPCs were isolated from human unbilical cord blood using methods of density gradient centrifugation combined with adherent culture. EPCs were induced to differentiate into endothelial cells under selective culture medium, which was identified by means of flow cytometry, immunofluorescence and in vitro angiogenesis. (2) EPCs were in vitro labeled with different concentrations of SPIO nanoparticles to observe labeling rate of SPIO and its effect on cellular viability of EPCs by trypan blue dye exclusion assay and MTT assay so as to select the optimal concentration of SPIO. (3) After establishment of glioma model in nude mice, the SPIO labeled EPCs were transplanted into nude mice by tail vein injection. The MRI was carried out at days 1, 3, 5 and 7 after transplantation and pathological tissues removed at corresponding time points. 1.5T MRI and Prussian blue staining of pathological specimens were used to study temporal-spatial distribution of EPCs in glioma model.Results:(1) EPCs isolated from umblical cord blood form colony. Under the selctive medium after 20 days of culture, EPCs express phenotype of mature endothelial cells, confirm it differentiate into mature endothelial cells. In the cytoplasm, transmission eletron microscope detect the Weibel-Palade body which is the characteristic structure of endothelial cells. The cells enrich mitochondria, endoplasmic reticulum and other cell organelles, suggesting a strong cell metabolism. Immunofluorescence assay positive represented EPCs differentiation into endothelial cells. Angiogenesis assay indirectly proved that the cultured cells had same fuction of endothelial cells. (2) Prussian blue staining showed numerous blue-stain particles in the cytoplasm of the SPIO-EPCs. Transmission electron microscopy detect a large number of high-density substances within cytoplasm. Higher concentration of SPIO enhance the label efficiency but resulted in greater cytotoxicity. When SPIO was at 25ug/ml and 50ug/ml, the trypan blue staining show proportion of dead cells for approximately 9%, 12% respectively, with no statistical difference compared with that unlabeled cells (approximately 10%). However, when SPIO was at concentration of 75ug/ml, 100ug/ml and 125ug/ml, the percentage of dead cells reached 21%, 33% and 49% respectively, significantly higher than that of unlabelled cells (approximately 10%) (P<0.05). MTT growth curve confirmed that the proliferation viability of EPCs showed no significant change when SPIO is at concentration less than 50ug/ml, but significantly reduced when the concentration of SPIO is higher than 75ug/ml. (3) At the first day after EPCs transplantation, hypointense signal detected by MRI scanning in glioma zone and became more clearly with time. There have no hypointense in the control group as well as in the contralateral normal brain tissue of experimental group. The area of hypointense signals on MRI is in accordance with Prussian blue stained cells in the histological section. The number of blue EPCs was significantly higher than the contralateral normal brain tissue and other organs (heart, liver, spleen, kidney and lung), the blue stained cells mainly gathered in the area of tumor microvascular active site.Conclusion:(1) The human umbilical cord blood is an ideal source for EPCs that can be induced to differentiate into endothelial cells directly under the conditional culture system. (2) EPCs can be easily and efficiently labeled by certain concentration of SPIO without interference on the cellular viability and proliferation. The labeled cells can be tracked by 1.5T MRI in vivo. The optimal concentration of SPIO is 25～50ug/ml. (3) EPCs may be imaging marker for early diagnosis of glioma and can be an effective vector for target molecular treatment of glioma. 1.5T MRI can track the migration, distribution and prognosis of transplanted cells in a temporal-spatial pattern. 1.5T MRI is an effective method for monitoring transplanted cells in vivo. |
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