Comparison Of BRDU And SPIO Labeling Of Hfbs

【Objective】:To test the physical and magnetic property of iron oxid nanoparticles in dextran-coated USPIO MRI contrast agent; to explore the efficiency of super-paramagnetic iron oxide (SPIO)and BrdU with different concentrations in vitro labelling human fibroblast cells, the influence on cellular viability and the characteristics of SPIO labelling HFBs in vitro magnetic resonance imaging, And to determine the optimal labelling agent for Hfbs labelling by comparing the above two methods and lay a foundation for further research on the distribution, migration, differentiation and prognosis of the transplanted cells.【Methods】:The dextran-coated iron oxide nanoparticles are obtained by means of co-precipitation method; the size and distribution of them is measured by transmission electron microscope; and the susceptibility is measured by vibrating sample magnetometer. Respectively mix SPIO-PLL compound of different concentrations with substrate (final concentrations of iron: 150μg/ml, 100μg/ml, 50μg/ml, 25μg/ml; final concentration of PLL: 7.5μg/ml). Cultivate it with Hfbs for 12h, 24h, 48h and 72h and collect the cells. Assess cellular viability with Trypan Blue Dye Exclusion method and cells labelling efficiency with Prussian Blue Stain method. Observe the positions of iron particles inside cells with transmission electron microscope and perform MR scanning to measure the signal intensity change of the cells. Respectively collect cells after cultivation with BrdU (final concentration: 5μmol/L, 10μmol/L and 15μmol/L) for 24h, 48h and 72h to test cellular viability with Trypan Blue Dye Exclusion method and labelling efficiency with immunocytochemistry staining. Compare those two motheds in terms of labelling time, labelling efficiency, cellular viability and shape.【Results】:The core of the sample obtained is iron oxide with core diameter of around 5nm, overall diameter of 20~35nm after dextran coating, relaxation rate of 0.155×106/(mol·sec), and quality saturation magnetic intensity of 69.42162emu/g Fe. Trypan Blue Dye Exclusion test shows the cellular viability difference between the cells labelled with 25μgFe/ml, 50μgFe/ml and the comparing groups is statistically insignificant (P>0.05); and the cellular viability difference between the cells labelled with 100μgFe/ml or higher concentration and the comparing groups are significant (P<0.05). Prussian Blue Stain test shows: the labelling efficiency of SPIO-PLL compound (95.2±3.92)% is obviously higher than that of pure SPIO (12.5±2.65) % (P<0.05); the labelling efficiency difference between 25μg Fe/ml, 50μg Fe/ml, 100μg Fe/ml and 150μg Fe/ml SPIO-PLL compound is statistically insignificant (P>0.05). The TEM shows that iron particles are concentrated inside the endosome and lysosome. MR scanning shows a gradual reduction of the labelled cells'signal intensity as the iron concentration increases; with 72h of labelling, cellular viability of different concentration BrdU is (94.5±1.5)%, cellular viability of the unlabelled cells is (98.2±1.8)%, with no obvious difference between the two (P>0.05). The labelling efficiency of 5μmol/L BrdU is (54.2±1.2)%, lower than that of 10μmol/L BrdU and higher BrdU concentration (79.87±1.52)% (P<0.05). There are no obvious differences between the group of 10μmol/L and 15μmol/L.【Conclusions】:The labelling efficiency of SPIO-PLL compound with concentration of 25μgFe/ml for 24h is obviously higher than that of BrdU with concentration of 5μmol/L. The two methods have no influence on cellular viability.