| Background and PurposeMalignant tumors, which are characterized by rapid growth, high morbidity and mortality, have become a major threat to human health. Tumor growth, metastasis and recurrence are inseparable from the formation of blood vessels. Although there is disordered structure for tumor tissue, the angiogenesis can play equally important role in the process when tumor and organs occurs from the view of angiogenesis. Endothelial progenitor cells (EPCs) are the precursors of vascular endothelial cells, and they have the potential to differentiate into the vascular endothelial cells. In the normal condition, the peripheral blood contains only small amounts of EPCs, and tumor tissues can release various cytokines to promote the recruitment of the EPCs from the bone marrow to the peripheral circulation. After that EPCs gradually aggregate to the active parts of angiogenesis, and then participate in the angiogenesis. This feature of EPCs has brought us new opportunities to treat cancer. New strategies of anti-cancer therapy are likely to present either by direct inhibition of the proliferation and differentiation of endothelial progenitor cell or utilizing it as a carrier, to carry tumor suppressor genes, and specifically enter into tumor in the future.In this study, the attachment growth method was used to isolate and culture rat bone marrow-derived endothelial progenitor cells. And poly-L-lysine (PLL) was used as a transfection agent to induct ultrasmall superparamagnetic iron oxide (USPIO) particles into endothelial progenitor cells in vitro, and then we studied the effect of magnetic markers on the biological characteristics of endothelial progenitor cells. Magnetic resonance imaging was performed to observe the labeled cells in vitro and look for the optimal sequence in vitro, and provide evidence for the following study on the transplanted cells migration and homing.Materials and methodsSprague–Dawley(SD) rats weighing about 100g were prepared and bilateral femur and tibia were taken off for marrow collection. Lymphocyte separation medium was used to isolate the mononuclear cells, and the attachment growth method was used to purify endothelial progenitor cells. Poly-lysine (PLL) was used as the transfection agent to induct ultrasmall superparamagnetic iron oxide (USPIO) particles into endothelial progenitor cells in vitro with the concentration of 25ug/ml. The positive labeling rate of cells was calculated 1, 3 and 5 days later, and cell vitality was measured as well. MTT assay was performed to evaluate the impact of USPIO-PLL on the proliferation capacity of cells. T2 and T2* maps were achieved at different concentrations of suspened cells to measure the relaxation time and the relaxation rate, and the correlation between the cell concentration and the relaxation rate was analyzed statistically.ResultsThe rat bone marrow mononuclear cells collected by density gradient centrifugation can differentiate into endothelial progenitor cells with the induction of the EGM-2 culture. USPIO-PLL, as an intracellular contrast agent, can label endothelial progenitor cells with high efficiency. Prussian blue staining method was used to label iron particles within the cytoplasm, and the labeling rate for 72h can reach as high as 98%. Trypan blue stain and MTT assay show that magnetic tag has no significant effect on the biological characteristics of cells. In vitro T2 and T2 * maps demonstrated that, the relaxation rate was decreased with the cell concentration and there is a linear correlation. The slope of R2 * effect is 3.58 times larger than that of R2 effect.ConclusionMononuclear cells can be induced and differentiated into endothelial progenitor cells in vitro. Using PLL as a transfection agent, USPIO can label EPCs effectively, and has no significant effect on stem cell growth at an appropriate concentration. Clinical application-oriented 3.0T MR T2 * map sequence is sensitive to the labeled cells and can demonstrate the concentration change of magnetically labeled cells in vitro. |
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