| Malignant tumors are common and frequently occuring diseases in human being, the key of increasing survive rate and improving living quality is to diagnose the malignant tumors in an early stage. The traditional imaging methods such as ultrasonography, computed tomography, magnetic resonance (MR) and so on usually aim to materiality mass, but the patients are already in the advanced stages, which shows a palliative curative effect and a poor prognosis. Therefore, seeking a new specific way to diagnosis tumor in early stage is an aim for all medical workers worldwide.The development of molecular imaging over the past decade has provided a novel visualization for early and non-invasion diagnosis of diseases, and has a tremendous perspection of apply and develop in diagnosis maliganat tumors in an early and specificity stage. In the research on molecular imaging, targeted probe with good specificity and high affinity is a critical factor in vitro and vivo molecular imaging.The molecular probe construsted by use antisense gene technique in molecular biology: we chose c-erbB2 oncogene as targeted gene and prepared the antisense oligodeoxynucleotide (ASODN) of complementary c-erbB2 oncogene in gene synthesis technique and labeled superparamagnetic iron oxide (SPIO) nanometer using chemical cross linking method, to produce the antisense probe. The basic group can complementarity and specifically combine with mRNA of oncogene when inducted in body, so it can distribution special in tumor tissue which have enlarge and over-expressive oncegene, and the location and size of tumor tissue can be demonstrated when magnetic resonance imaging. If ASODN, which has opposited sequence with cellular DNA and mRNA sequence, may induct in cells, it can specifically combine with corresponding DNA and mRNA to increase DNA and mRNA concentration. It is obvious that antisense gene imaging technique can detecting the tumor tissue which only has enlarge and over-expressive and no noumenal, so it can diagnosis tumor in early state.In this experiment, we chose c-erbB2 oncogene as targeted gene because c-erbB2 oncogene is homologous gene of neu gene in human being, which amplify and expression only in malignant tumors. Moreover, c-erbB2 oncogene have close correlation with pathologic grade, lymph node metastasis and clinic stage of tumor, and frequently use as a major index of clinic chemical treatment plan and prognosis judgments. Therefore, this gene provides a good situs for targeting diagnosis, once the amplification and over-expression of c-erbB2 oncogene can cause cellular mRNA increase and provide more conjunctive targeting situs. Because eugonic tumor cell membranes, have many transferrin receptors which can guide SPIO-labeled antisense probe into tumor cells, hence, the paramagnetism materials increase. After MR scanning, will get the imaging with clarity anatomic structure, high noise-signal ratio and well contrast is obtained, so oncogene specificity expression may be displayed from imaging.MR is ideal molecular imaging detecting equipment which has long imaging time widow and high temporal and spatial resolutions and good picture contrast. However, the most disadvantage of MR is lower sensitivity in detecting signal, and this method only measureμmol grade paramagnetism material in tissues. To achieve visualized detection of oncogene expression, we must label paramagnetism material in key molecular in tumor formation. SPIO, the frequently used paramagnetism material, has magnetism aquare. SPIO can arrangement freely along magnetic field which cause tremendous microcosmic phase difference in periphery proton, which resulting in the protonic T2 desphase relaxation quickening, the signal of tissue's T2 weight imaging reducing distinctly and signal difference between tissues increasing distinctly. Its magnetism disappear rapidly when the magnetic field is taken out. Therefore, it is a superparamagnetism. In addition, SPIO, which has lower toxin and less side effects, is an ideal and safety contrast medium and molecular carrier, and thus extensively used in tracing vivo stem cells (such as nerve stem cell, embry stem cell and mesenchymal tissue stem cell). However, most SPIOs accumulate in endothelial system such as liver, spleen and lymph node when it is injected directly in vivo. Therefore, they have low targeted effects to other organs and tumors. How to initiative targeting imaging to organs becomes a focus recently. Some study had achieved to mark large molecules, such as monoclonal antibody, specific proteins and plasmid DNA. To our knowledge, there has been little report on labeling ASODN by using SPIO。METHODS:Part 1: Preparation and test of SPIOThere are some commercial SPIO production abroad at present, but this production aren't purchased in domestic market and the price very costliness, the diameter from several nanometers to hundreds nanometers, the surfactant used are very difference, so the disadvantage to bring forth new ideas research which have knowledge property right. We preparation SPIO in nanometers grade with coprecipition method, and emphasis evaluation the token, magnetic parameter, acute toxicity and stability of SPIO sample, can be further study as gene carrier to us in MR gene imaging.1.The structure of SPIO is analyzed by X-ray powder diffraction method, its size is measured by transmission electron microscope and atomic force microscopy.2.The saturated magnetization, retentivity and relaxivity is measured by vibrating sample magnetometer and 1.5T MR system.3.Acute toxicity to mice of SPIO: 90 mice were divided into oral administration (n=30), intravenous injection (n=30) and intaperitoneal injection (n=30) groups randomly according to the administration pattern and doses. The oral administration with a total dose 2104.8 mg/kg and a volume 40ml/kg, intravenous with a total dose 438.5mg/kg and a volume 25ml/kg, intraperitoneal injection with a total dose 1578.6mg/kg and a volume 30ml/kg respectively, and 10 mice in each groups were used as the control group as the same administration pattern and the same doses (n=10). The general condition, mostly biochemical index and the main viscera pathological morphology were recorded in 14 days feeding period.4.According to the basic request of injection, the strengthen experimention and long-term experimention had done, the character, pH, iron content, size, saturated magnetization and T2 relaxivity were observed and determined.Part 2: Preparation and the quality test of antisense oligodeoxynucleotide probe labeled with magnetism1.Aim at transcribe start bit 5'-end of c-erbB2, and aritificial synthesis antisense oligodeoxynucleotide segment and corresponding sense oligodeoxynucleotide segment and non-sense oligodeoxynucleotide segment, the SPIO-labeled oligodeoxynucleotide segment was prepared through SPIO conjugated to ASODN using chemical cross linking method.2.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.On the basis of author's prophase experimental datum, the maximum dose were used in this experiment. Health and clean grade of 60 Kunming mice were divided into oral administration, intravenous injection and intaperitoneal injection groups randomly. The oral administration with a total dose 2104.8 mg/kg and a volume 40ml/kg, intravenous with a total dose 438.5mg/kg and a volume 25ml/kg, intraperitoneal injection with a total dose 1578.6mg/kg and a volume 30ml/kg respectively, and 20 mice in each groups were used as the control group as the same administration pattern and the same doses. All experimental mice were feed in 25℃, the general condition, blood routine examination and mostly biochemical index and the main viscera pathological morphology were recorded in 14 days feeding period.4.Observe the stability after placement: The character, pH, iron content, size, saturated magnetization and T2 relaxivity were observed and determined after 3 month placed in refrigerator at 4℃±1℃.Part 3: Pharmacokonetics parameters in rabbits and distribution and excretion in mice of antisense oligodeoxynucleotide probe labeled with magnetism1.Pharmacokonetics in rabbits: 15 rabbits, its body weight were 2.0±0.4kg, divided randomly into 3 groups. The antisense probes were injected at 3mg/kgFe (small dose), 6mg/kgFe (middle dose) and 12mg/kgFe (large dose) from left aures vein respectively, the vein blood 0.5ml collected from right aures vein at before injection and 1min,3min,5min,10min,20min,40min,60min,120min,180min,360min,720min after injection. The Fe concentration mensurated by atomic absorption spectroscopy method, the half-life period, area under curve (AUC) and clearance (CL), plasma protein combine ratio were calculated by 3P97 program automatically software curve equation.2.Distribution in mice: 35 BALB/c mice divided into randomly 7 groups, antisense probe at 12ml/kgFe injected from tail vein, MR scanning at 1min,10min,30min,60min,180min,360min after injection, the signal intension mensurated in liver, spleen, lung, kindey and muscle. After that, vein blood collected from orbit vein, the Fe content mensurated in liver, spleen, lung, kindey and muscle after put these mice to death through cervical spine dislocation. 3.Excretion in mice: 5 BALB/c mice breeding in metabolize cage, its urine and dejection collected before 24h injecte antisense probe, Fe content were determine as background. After that, antisense probe injected at 3.76ml/kgFe from tail vein, the Fe content were determined after injecte 0～24h and 24～48h.Part 4: SK-Br3 oncocytes which high expressive c-erbB2 oncogene were transfected in vitro use the antisense oligodeoxynucleotide probe1.Biology characteristic observated after SK-Br3 oncocytes transfected with antisense oligodeoxynucleotide: 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.2.The specificity observed after SK-Br3 oncocytes transfected: 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. To determine it specificity, the Fuda oncocytes which no-expressive c-erbB oncogene transfected with antisense probe, and SK-Br3 oncocytes transfected with sense probe and nonsense probe, SK-Br3 oncocytes transfected with SPIO and ASODN as control groups.3.In vitro MR imaging: transfected cells with antisense probe was placed into Ependoff tubes, which were fixed with 3 inches surface coil, and the Fuda oncocytes transfected with antisense probe, and SK-Br3 oncocytes transfected by sense probe and nonsense probe probe, SK-Br3 oncocytes transfected by SPIO and ASODN and distilled water as control samples. MR transection scanning was performed in all 7 samples by using 1.5T instrument. 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.Part 5: In vivo MR gene imaging of antisense oligodeoxynucleotide probe labeled by SPIO1.Set nude mice with xenografted tumors models: execute SK-Br3 oncocytes at logarithmic growth phase, oncocytes inoculation to right back limb use 30 nude mice with 5～6 week and 12g～18g weight, the nude mice with xenografted tumors models set successful after about 15 days.2.The dose of antisense probe and MR scanning time confirmed: according to pre-experiment and drug-time distributional curve graph of antisense probe in rabbit, 12ml/kgFe was confirmed in the vivo bearing nude mice MR scanning, the scanning time were confirmed at 10min, 30min, 60min, 180min and 360min after the probe injected, respectively.3.MR scanning sequence and parametes:①T1WI SE sequence TR/TE was 400/13 ms,excitation 6;②T2 WI FSE sequence TR/TE was 2500/40 ms,excitation 2;③T2* WI SE sequence TR/TE was 2000/80ms,excitation 2;④T2* FSPGR sequence TR/TE 130/9,excitation 2,a flip angle of 6°. The transection and coronal position were performed with section thickness of 4 mm, gap 1mm and a matrix of 256×222,and a field of view of 10cm.4.MR data analysis:The signal intensity of liver, spleen, heart, kindey, muscle and tumor in all bearing nude mice observated, the time-signal curve determined after the signal noise ratio mensurated.5.Pathology analysis: The nude executed and the liver, spleen, heart, kidney, muscle and tumor took out after scanning at each time performed, the sample staining by HE and prussian blue, observed in optical microscope.RESULTS:Part 1: Preparation and test of SPIO1.The superparamagnetic iron oxide nanoparticles coated with dextran was obtained by means of coprecipitation method, its component was iron oxide crystal. Transmission electron microscopy showed that the nucleus of iron oxide was mainly spherical and well dispersed, the diameter was no more than 5 nm and the size of the coated particles could not be revealed. But the AFM suggested that the dextran-coated SPIO was cubic and had good dispersion, the diameter of nucleus was less than 5 nm, and the coated particle was 20～35 nm in size.2.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.3.During the observation period, there were no any mice death, there were no significant different of the biochemical index in experimental group and control group, and no edema, degeneration and necrosis were seen in liver, spleen, kidney, heart, lungs by HE staining and marrow Wright staining, only a few blue particles were observed in liver and spleen in administration groups by Prussian blue staining, no in control group.4.The character, pH, iron content, size, saturated magnetization and T2 relaxivity of the samples after experimention were no statistics differences than that of before experimention under the condition of strengthen experimention and long-term experimention.Part 2: Preparation and the quality test of antisense oligodeoxynucleotide probe labeled with magnetism1.Chemical constitution of 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.2.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 0.156×106mo l-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.During the observation period, there were no any mice'death, only a few had appetite loss and increased drinking-water, diarrhea, sleep, increased activities and all abnormalities disappered in 3 days. There were no abnormality in color and morphology of liver, spleen, kindey, heart, lungs and bone marrow, none statistical differential of viscera coefficient. All cells of liver, spleen, kindey, heart, lungs and bone marrow had not edema, degeneration and necrosis either in HE or in Wright staining. There were no significant different of the blood routine examination and biochemical index in experimental groups and control group.5.After experimention, the character, pH, iron content, size, saturated magnetization and T2 relaxivity of the samples were no statistics differences than that of before experimention under the condition of placement in refrigerator at 4±1℃and 3 month.Part 3: Pharmacokonetics parameters in rabbits and distribution and excretion in mice of antisense oligodeoxynucleotide probe labeled with magnetism 1.Pharmacokonetics distribution coincide with two compartment model after administration the antisense probe at small dose, middle dose and large dose.2.The t1/2 was 38.96min, AUC was 7029.03ug·ml-1·min-1, CL 0.000427mg·kg-1·min-1 and plasma protein combine ratio 23.6% after administration at small dose.3.The t1/2 was 142.05min, AUC was 13502.21ug·ml-1·min-1, CL 0.000444mg·kg-1·min-1 and plasma protein combine ratio 24.1% after administration at middle dose.4.The t1/2 was 210.66min, AUC was 28311.73ug·ml-1·min-1, CL 0.000444mg·kg-1·min-1 and plasma protein combine ratio 24.3% after administration at middle dose.5.Atomic absorption spectroscopy method show that the antisense probe distribute mainly in liver and spleen, the peak time at 180min after injected, and came down slowly. Through mensurate the noise-signal ratio of some viscera with MR scanning, the similar conclusion had get.6.The output dose of the antisense probe pass by intestinal tract were more than that of kindey.Part 4: SK-Br3 oncocytes which high expressive c-erbB2 oncogene were transfected in vitro use the antisense oligodeoxynucleotide probe1. There were no significancant difference of proliferate activity in SK-Br3 oncocytes transfected by antisense probe detected with MTT method and trypan blue staining.2.There were inhibitory action of mRNA and protein expression in SK-Br3 oncocytes transfected by antisense probe detected with RT-PCR and Western blot, compare with control group (p<0.05).3.The morphology observation using optical microscope and eletron microscope show that some iron particles in SK-Br3 oncocytes transfected by antisense probe, no iron particles in control group.4.Compared with control group, the cellular iron content increase obviously in SK-Br3 oncocytes transfected by antisense probe mensurated by atomic absorption spectroscopy, there were significancant difference(F=198.79,P=0.00073).5.The signal decreased obviously in SK-Br3 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 (P<0.01).Part 5: In vivo MR gene imaging of antisense oligodeoxynucleotide probe labeled by SPIO1.The bearing tumor nude mice models were set up successfully, in outlook the two models were no difference.2. T1WI SE sequence show that the change of signal intension were not sensitivity after the antisense probe injected at various time. But the other three sequences (T2WI FSE sequence,T2* WI SE sequence and T2* FSPGR sequence)show that the signal intension decrease after the antisense probe injected at various time, there were difference on signal noise ratio.3.The signal noise ratio decreased after antisense probe injected 10min of tumor in SK-Br3 bearing nude mice model, but the decrease can not confirm use naked eye, the signal decrease lightly at 30min and decrease distinctly and minimum at 180min, rise up gradually after that. The signal noise ratio decreased after antisense probe injected 10min of tumor in Fuda bearing nude mice model, but the decrease can not confirm use naked eye, and the signal noise ratio were not decrease, and no difference at statistics.4.Pathology show that there were a lot of spot blue iron grain in tumor tissue of SK-Br3 nude model and no blue iron grain in tumor tissue of Fuda nude model.CONCLUSION:Firstly, the probe carrier (SPIO) preparted by coprecipitation, SPIO had small size, well dispersity and ideal magnetism parameter, and acute atoxicity, well stability, so the SPIO was provided with molecular probe carrier and MR imaging contrast agent.Secondly, the antisense probe was obtained by chemical cross-linking approach,namely labeled SPIO with ASODN. The probe had high link rate, small size, superparamagnetism and acute atoxicity to mice, and the stability kept after placed in 4±1℃3 months. Its had longer half life, and moderate plasma protein comnine rate. 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. The probe had better targeting after used in vivo MR imaging on nude mice with xenografted tumors models.The antisense gene imaging which in molecular imaging can find out tumor's occurrence and development process objectively in extremely early stage (gene level) before appear clinical symptom, is a prospectly new method to diagnosis tumor specificityly and has widely application.
|
PDF Full Text Request