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Purpose (1) to observe the growth of rat breast cancer differentiation and pathological features of lymphangiogenesis; (2) the synthetic targeting the podoplanin the MRI contrast agent of gold magnetic particles, in vitro observation of the physical and chemical properties of nanoparticle contrast agents; (3) Podoplanin molecular targeted MRI in vivo imaging, evaluation of breast cancer lymphangiogenesis. Gold magnetic particles (GoldMag-Coreshell, GoldMag) method (1) the use of both magnetic particles superparamagnetic colloidal gold conjugate biomolecules performance chelating anti-podoplanin antibody synthesis the targeting the podoplanin the MRI GoldMag particulate contrast agent ( PodAb-GoldMag), chelating non-specific IgG synthesis of the MRI IgG-containing nanoparticles (IgG-GoldMag) control chelate around the two antibodies was measured at 280nm absorbance values ??calculated the antibodies in GoldMag surface coupling efficiency; ELISA double antibody sandwich method for the determination the nanoparticles of immune activity. (2) with a laser particle size analyzer respectively on GoldMag core particles ultra-small superparamagnetic iron oxide (USPIO) GoldMag IgG-GoldMag of PodoAb-GoldMag, Zeta potential measurement; transmission electron microscope observation of USPIO, GoldMag and PodoAb-GoldMag nanoparticles morphological changes and the measured particle size variation. (3) with phosphate buffered saline (PBS) as a diluent USPIO and GoldMag were diluted to 0.5 ~ 1000μg/ml 47 different concentrations with PBS solution as control, received a total of 48 concentration, row FSE T1WI, FSE T2WI and GRE T2 * WI magnetic resonance imaging. Observation the different concentrations USPIO and GoldMag of signal strength variation in the sequence of three kinds, and to calculate the ratio of signal intensity changes, draw the concentration - the graph of the signal strength. (4) 80 Wistar female rats, DMBA gavage (DMBA)-induced mammary tumors, 75 rats were successfully induced mammary tumor, were randomly divided into A, B, C, D 4 groups, group A (PodoAb -GoldMag group) 30 rats, the B group (IgG-GoldMag group), the C group the (GoldMag group) and Group D (Gd-DTPA group) of 15 rats. 1.5 T magnetic resonance imaging system FSE T1WI, T2WI, fat suppression sequences, diffusion-weighted imaging, A, B, C group, respectively, prior to the injection of contrast agent, immediately after contrast injection, 5 min, 15 min, 30 min, 1 h and 2 h-line breast lumps GRE T2 * WI enhanced scan, D group phase point line FSE T1WI enhanced scan at the same time, measuring the signal strength, draw dynamic contrast-enhanced scan time - signal intensity curve. (5) After the scan, perfusion fixed specimens, same breast lumps are drawn from the central area and the surrounding areas, HE staining and podoplanin antigen immunohistochemistry, immunofluorescence staining and Prussian blue iron stain, observation of breast tumor pathological type and lymphangiogenesis same tumor lymphangiogenesis were observed two different parts of the Central District and surrounding areas. (6) breast tumor HE staining and immunohistochemical staining, and LVD check results with the MRI image control analysis, analysis of the MRI findings of breast tumor pathology basis to discuss the effect of podoplanin molecular targeted MRI evaluation of in vivo imaging of breast cancer lymphangiogenesis. Results (1) successful chelation anti-podoplanin antibody with GoldMag of the synthesis the target the podoplanin The PodAb-GoldMag, anti podoplanin antibody and GoldMag, average binding rate of 64.81%, 43% anti-podoplanin antibody immune activity. (2) USPIO the Zeta potential of 12.0 ± 18.9 mv, particle size distribution of 15 to 30 nm; Zeta potential of GoldMag to 25.2 ± 95.2 mv diameters range from 40 to 80 nm; The IgG-GoldMag the Zeta potential of 18.2 ± the 1.6 mv PodAb-GoldMag the Zeta potential was 25.5 ± 20.8 mv, particle size distribution range of 80 to 100 nm. (3) GoldMag with USPIO MRI signal variation of the basic different imaging sequence signal strength compared with the concentration increase FSE on T1WI was signal strength tended to increase FSE T2WI signal intensity decreased slightly, GRE T2 * WI signal intensity decreased . Comparison of signal strength of the two contrast agents, the signal strength in the FSE T1WI, concentrations higher than 30μg/ml there are significant differences (P LT; 0.05) FSE T2WI, a concentration of 40 ~ 90μg/ml signal strength when there are significant differences (P lt; 0.05), GRE T2 * WI, the signal strength of the two contrast agents no significant difference (P gt; 0.05). (4) 75 rats successfully induced breast lumps, 68 rats induced a breast lump, seven rats induced two breast lumps. 75 rats, the proliferative breast disease 21, two inflammatory granuloma, 52 breast cancer, breast lumps evoked a success rate of 93.8%, breast cancer induced a success rate of 69.3%. (5) in the plain MRI scan T1WI, benign and malignant lesions showed low signal or signals; performance for high signal and signal-based mixed signal in T2WI, benign breast disease and breast malignancies, 42 performance mainly for high signal and other signals mixed signal, and the remaining 10 performance for low signal-based mixed signal; fat suppression sequences T2WI the mammary fat high signal is suppressed, benign and malignant lesions showed high signal. (6) GoldMag targeting enhanced scan, A, B, C 3 breast cancer signal strength in contrast injection immediately decreased significantly (P lt; 0.01), and rise gradually over time, 1 h after injection, the signal strength is no longer changes in group A signal strength than before the injection is low, the differences were significant sex (P lt; 0.05), basic groups B, C, signal strength back to the level before the injection, no significant difference in sex (P gt; 0.05). Different histological types of breast cancer signal strength before and after contrast injection without significant differences (P gt; 0.05), the signal strength in different degree of differentiation of breast cancer contrast injection immediately to within 5min difference was significant (P lt; 0.05), other was no significant difference in the point-in-time. (7) LVD with the decrease of the degree of differentiation of breast cancer was significantly higher (P lt; 0.05), the LVD and A group of breast tumor signal to reduce the degree was a significant negative correlation (r = -0.814, P = 0.000), and B, C two reduce the signal strength of the group of breast cancer showed no significant correlation (P gt; 0.05), and group D breast tumor signal intensity increased the degree was a significant positive correlation (r = 0.972, P = 0.000). Conclusion (1) GoldMag superparamagnetic composite particles as a core-shell structure, its core USPIO USPIO GoldMag MRI signal characteristics similar, with increasing concentration, FSE on T1WI was signal strength tended to increase, FSE T2WI signal intensity slightly downward GRE T2 * WI signal intensity decreased. GRE T2 * WI the USPIO and GoldMag MRI imaging sequences. (2) of successful chelation targeted PodAb-GoldMag nanoparticle contrast agents, anti-podoplanin antibody and GoldMag, average binding rate was 64.81%, the anti-podoplanin antibody immune activity of 43%, PodAb-GoldMag the Zeta potential for 25.5 ± 20.8 mv particle size distribution range of 80 ~ 100 nm. (3) rats induced breast MRI Ping sweep of T1WI performance for low signal or signal; the T2WI majority showed high signal and signal-based mixed-signal; fat suppression sequences T2WI lesions showed high signal with human breast cancer MRI findings similar. (4) proliferative breast disease seen only a little lymphangiogenesis and a small amount of the larger, more mature lymphatic vessels. Breast cancer lymphangiogenesis multi stromal tumor edges around the cancer nests, LVD increased with the degree of differentiation of breast cancer. (5) PodAb-GoldMag enhanced scan the surrounding area of ??breast cancer signal strength significantly lower than the central area; different degree of differentiation and different histological types of breast cancer, PodAb-GoldMag enhanced scan signal intensity changes basically similar. (6) PodAb-GoldMag targeting enhanced MRI and tumor lymphatic microvessel density and enhanced scan, according to the rat mammary tumor PodAb-GoldMag preliminary identification of its lymphangiogenesis vivo assessment of breast cancer lymphangiogenesis.
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