Antisense Probe For C-erbB2 Oncogene And Magnetic Resonance Gene Imaging

Objective :The goals of this study were to image oncogene c-erbB2 using SPIO-radiolabelled antisense oligodeoxy- nucleotides(ASODN) to bind complementally target gene to diagnose cancer. To establish tumor oncogene imaging new method ,that has such characters as higher sensibility, higher selectivity and higher target-tropism.Method:1.Preparation and test of SPIO: the iron oxide nanoparticles is obtained by means of Co-precipitation method .The structure of SPIO is analyzed by X-ray powder diffraction method, its size is measured by transmission electron microscope and atomic force microscopy.The saturated magnetization, retentivity and relaxivity is measured by vibrating sample magnetometer and 1.5T MR system2.Preparation and test of antisense oligodeoxynucleotide probe labeled with magnetism:Aim at transcribe start bit 5'-end of c-erbB2, and aritificial synthesis antisense oligodeoxynucleotide segment, the SPIO-labeled oligodeoxynucleotide segment was prepared through SPIO conjugated to ASODN using chemical cross linking method, its configuration was detected by atomic force microscope, the conjugating rate and biology activation were determined by high performance liquid chromatography, the stability was detected by polyacrylamide gel electrophoresis. the T2 relaxivity was determinate with a 1.5T MR system and the magnetization parameters were determined by vibrating-sample magnetometer.3.Biology characteristic observated after SK-Br-3 oncocytes transfected with antisense oligodeoxynucleotide and in vitro MR imaging:cellular activity determined by MTT method and Trypan Blue staining; mRNA expressive of c-erbB2 oncogene by RT-PCR semi-quantitative analysis; protein expressive of c-erbB2 oncogene by Western blot,some means as Fe content in cells mensuration by atomic absorption spectroscopy method, and Fe distribution observation by prussian blue cell stain and transmission electron microscope,Collecting sequence was GRE sequence, flip angle 30°TR/TE 225/5.3ms, field of view 16.0mm, slice thick 1.8cm, matrix 256×12⁸.Result:1.The superparamagnetic iron oxide nanoparticles coated with dextran was obtained by means of coprecipitation method, its component was iron oxide crystal. Transmission electron microscopy and AFM showed that the nucleus of iron oxide was mainly spherical and well dispersed, the diameter was no more than 5 nm,and the dextran-coated SPIO was cubic and had good dispersion, the coated particle was 20～35 nm in size;the sample's T2 relaxivity was 0.155×106mo l-1·sec-1, and the saturated magnetization and specific saturated magnetization and specific retentivity and retentivity were 69.42162emu/g Fe, 68.39568emu/g, 30.4648emu/g and 21.46374Gs, respectively, the magneticzatic curve showes that the sample exhibit superparamagnetism. The magnetic effect of the sample similar with that of the foreign producation recorded by literature.2. c-erbB2 oncogene antisense probes can be observed by atomic force microscopy. The probes with diameter of 25-40nm are in order, uniformity and arrangement rule, it can be separated each other and appear cube with rugged surface morphology;the conjugating rate was 99% and had inhere biological activity were determined by high performance liquid chromatography, and had well-stability detected by polyacrylamide gel electrophoresis.3. Its T2 relaxivity was 0.156×10⁶mol^-1·sec^-1 determinate with a 1.5T MR system and the saturated magnetization and specific saturated magnetization and specific retentivity and retentivity were 69.4238emu/g Fe, 68.4134emu/g, 30.3541emu/g and 19.7345Gs, respectively,which determined by vibrating-sample magnetometer.4. There were no significancant difference of proliferate activity in SK-Br-3 oncocytes transfected by antisense probe detected with MTT method and trypan blue staining;There were inhibitory action of mRNA and protein expression in SK-Br-3 oncocytes transfected by antisense probe detected with RT-PCR and Western blot, compare with control group5. The morphology observation using optical microscope and eletron microscope show that some iron particles in SK-Br-3 oncocytes transfected by antisense probe, no iron particles in control group.6. The signal decreased obviously in SK-Br-3 oncocytes transfected by antisense probe mensurated by MR cellular imaging , there were significancant difference of the signal to noise ratio compared with the control groups .Conclusion:1.The probe carrier (SPIO) preparted by coprecipitation, SPIO had small size, well dispersity and ideal magnetism parameter, so the SPIO was provided with molecular probe carrier and MR imaging contrast agent.2.The antisense probe was obtained by chemical cross-linking approach,namely labeled SPIO with ASODN. The probe had high link rate, small size, superparamagnetism.3.The viabliity and proliferation of SK-Br-3 was not affected after transfected by the probe, and the probe had more specificity and can enter the intracellulare efficiency, the signal intensity of SK-Br-3 decrease distinctly.