ABSTRACT
This study was undertaken to explore and compare the radiochemical behavior and biological property of antisense oligonucleotide (ASON) labeled with Technetium-99m using two methods: N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycline (NHS-MAG3) versus hydrazino nicotinamide derivative (SHNH). After SHNH and NHS-MAG3 were synthesized, ASON was labeled with Technetium-99m using SHNH and NHS-MAG3 as a bifunctional chelator, separately. The stability in vivo and in vitro, the combination with plasma albumen of rabbit, the biodistribution in BALB/ C mice and the HT29 cellular uptake were compared between labeled compound 99mTc-SHNH-ASON, using SHNH as a bifunctional complex reagent, and 99mTc-MAG3-ASON, using NHS-MAG3 as a bifunctional chelator. The results revealed that the labeling rate and the stability of 99mTc-MAG3-ASON were evidently higher than that of 99mTc-SHNH-ASON (P < 0.05), the combination rate of 99mTc-MAG3-ASON with plasma albumen was markedly lower than that of 99mTc-SHNH-ASON (P < 0.05); the biodistribution of 99mTc-MAG3-ASON was markedly lower than that of 99mTc-SHNH-ASON in blood, heart, stomach and intestines (P < 0.05), slightly lower than that of 99mTc-SHNH-ASON in liver and spleen (P > 0.05), and markedly higher than that of 99mTc-SHNH-ASON in kidney (P < 0.05); the HT29 cellular uptake rates of 99mTc-MAG3-ASON was markedly higher than that of 99mTc-SHNH-ASON (P < 0.05). Therefore, the radiochemical behavior and biological property of 99mTc-MAG3-ASON labeled using NHS-MAG3 is better than that of 99mTc-SHNH-ASON labeled using SHNH.
Subject(s)
Animals , Humans , Mice , Colonic Neoplasms , Metabolism , Pathology , Glycine , Chemistry , Pharmacokinetics , Isotope Labeling , Methods , Mice, Inbred BALB C , Niacinamide , Chemistry , Pharmacokinetics , Oligonucleotides, Antisense , Chemistry , Pharmacokinetics , Radiopharmaceuticals , Pharmacokinetics , Succinimides , Chemistry , Pharmacokinetics , Technetium Tc 99m Mertiatide , Chemistry , Pharmacokinetics , Tumor Cells, CulturedABSTRACT
This study was conducted to screen the endocrine exophthalmos-associated genes or cDNA fragments and provide a basis for exploring the pathogenesis. The cDNA from the thyroid tissues of patients with hyperthyroidism and endocrine exophthalmos (HT&EE) was used as tester cDNA, and that from the thyroid tissues of patients with HT but free from EE was used as driver cDNA. The subtractive PCR products between tester and driver cDNA were obtained through two cycles of subtraction hybridization and two cycles of PCR by using suppression subtractive hybridization, and then were inserted into pT-Adv, a cloning vector. The ligated DNAs were transformed into E. coli DH5alpha competent cells and incubated for proper blue/white color development. Forty-eight white colonies were randomly picked and their inserts were colony PCR amplified. The PCR product from one of the colonies contained two inserts; the others contained single insert, having a size of 0.2 kb to 2 kb. The inserts of transformants were arrayed on nylon membrane. After cDNA/Rsa I digestion the thyroid tissues of patients with HT, and of patients with HT&EE, were labeled with digoxigenin; the nylon membranes were then hybridized respectively with the two cDNA probes for a high throughput screening for positive clones. The clones which were hybridized with the cDNA probe of HE&EE patients but not hybridized with the probe of the HT patients or showed only faint signal of hybridization, were chosen as positive clones and their inserts were candidates for the endocrine exophthalmos genes or cDNA fragments. About 50% of the clones were confirmed as positive clones. The cDNA fragments in the positive clones were the endocrine exophthalmos-associated genes or cDNA fragments. Endocrine exophthalmos
Subject(s)
Humans , Cloning, Molecular , DNA, Complementary , Genetics , Graves Disease , Genetics , Polymerase Chain ReactionABSTRACT
Graves' ophthalmopathy (GO) is also called thyroid-related eye disease, infiltrative ophthalmopathy, which is related with the autoimmunity of thyroid, especially hyperthyroidism. Its morbidity ragnes from five percent to ten percent of hyperthyroidism, and the morbidity of male patients is higher than that of the female patients. The treatment of severe GO is a difficult task for doctors. The therapeutic effect is not always satisfactory. In order to solve this knotty problem, researchers have been devoting themselves to the development of new therapeutic methods. Here, the development of the therapies for GO is introduced, and the trends of treatments are prospected.
Subject(s)
Humans , Combined Modality Therapy , Exophthalmos , Radiotherapy , Therapeutics , Graves Disease , Radiotherapy , Therapeutics , Immunoglobulins , Therapeutic Uses , Prednisolone , Therapeutic UsesABSTRACT
To explore the preparation method of liposome-mediated 99m-technetium-labeled antisense oligonucleotides of c-myc mRNA and lay foundations for antisense imaging and treatment, antisense oligonucleotides (DNA) with 15 bases and di-functional chelate, hydrazino nicotinamide derivatives, were synthesized. After DNA combined with chelate, they were labeled with 99m-technetium to form compounds, 99mTc-chelate-DNA (99mTc-DNA), and were purified through Sep-Pak reverse column(C18, Waters) by using methanol and water as eluent. The leaching curve was made; the labeling efficiency was calculated. The products were then encapsulated with cation liposome to form liposome-mediated 99mTc-DNA. The radiochemical purity and stability of the liposome-mediated 99mTc-DNA were tested through strip chromatography. The labeling efficiency was 63.37% +/- 3.51% at the radioactive concentration of 1480 MBq, 62.52% +/- 3.69% at that of 740 MBq, 59.82% +/- 5.12% at that of 592 MBq. There were no significant differences between these labeling efficiencies. The radiochemical purity was 96.47% +/- 3.01%. The liposome-mediated radiolabeled antisense oligonucleotides were stable after incubation with water or serum. Therefore liposome-mediated radiolabeled antisense oligonucleotides could be obtained through hydrazino nicotinamide derivatives as di-functional chelate and liposome as vector.
Subject(s)
DNA, Antisense , Drug Compounding , Methods , Drug Stability , Isotope Labeling , Methods , Liposomes , Proto-Oncogene Proteins c-myc , Genetics , RNA, Messenger , Genetics , TechnetiumABSTRACT
The purpose of this study was to evaluate the efficacy of immunosuppressive agents, 99Tc-MDP and both of them in treating patients with Graves' ophthalmopathy(GO). The efficacy was evaluated by randomized controlled trial involving a total of 66 patients. In 22 patients treated with immunosuppressive agents, the general efficacy rate was 19/22, the incidence rate of serious side-effect was 8/22. In 20 patients treated with 99Tc-MDP, the general efficacy rate was 17/20, the incidence rate of serious side-effect was 2/20. In 24 patients treated with immunosuppressive agents and 99Tc-MDP, the general efficacy rate was 22/24, the incidence rate of serious side-effect was 2/24. The results suggested that in the treatment of Graves' ophthalmopathy, when satisfactory efficacy was obtained, the serious side-effect and 'rebound' of symptom could be avoided by using immunosuppressive agents in combination with 99Tc-MDP.