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Objective Through detecting the expression of chemokine CCL21 and its receptor CCR7 in thyroiditis of NOD mice induced by excessive iodine and thyroglobulin (Tg) immunization,to explore the mechanism of lymphocytic infiltration.Methods Seven to eight weeks old female NOD (non-obese diabetic) mice were used,the mice were divided into four groups by random number table,7 mice for each group.Control group:intoke distilled water;,Tg group:intoke'distilled water,use pig Tg emulsified in Freund's complete adjuvant subcutaneous injection to immunization,0.10 mg Tg for each mouse at 8 weeks old,strengthened immunity at 11 and 15 weeks old (0.05 mg Tg for each mouse,and emulsified by Freund's incomplete adjuvant); iodine excess (HI) group:intoke 0.05％ NaI water; iodine excess combined with Tg (HI + Tg) group:intoke 0.05％ NaI water,Tg immunization method was same to Tg group; all mice were raised in specific pathogen free (SPF) environment.All mice were executed at 16 weeks old,and removed the thyroid tissue.Morphologic change in thyroid was observed by HE staining.Immunofluorescence (IF) staining was used to observe CD3,CD22,CCL21 and CCR7 expression in thyroid.Real-time PCR was used to detect CCL21 mRNA expression in thyroid.Results The morphologic change of thyroid was observed under the light microscope,in HI group,thyroid follicle distended,colloid accumulation,lymphocytes accumulate together and disorganization was seen; Tg group thyroid had scatted lymphocytes; HI + Tg group enhanced inflammation response and disorganization.IF staining showed that lymphocytes infiltrated in thyroid were almost CD3+ T cells,only few scatted CD22+ B cells; CCL21 and CCR7 were negative in control group,CCR7 positive cells were scatted in Tg group but no CCL21 expression,in HI and HI + Tg groups,we could see CCL21 strong positive expression in inflammation area and also had CCR7 positive cells there.CCL21 mRNA relative expression in thyroid:Tg group (3.20 ± 0.18),HI group (1.72 ± 0.31),and HI ± Tg group (9.94 ± 0.32) increased compared to control group (1.00 ± 0.17,all P ＜ 0.01).Conclusion CCL21 and its receptor CCR7 have improved lymphocytic infiltration in autoimmune thyroiditis
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AIM: To investigate the effects of poly (I:C) as virus mimics on iodine excess-induced chronic lymphocytic thyroiditis in NOD mouse. METHODS: Female, 32 NOD mice were randomly divided into 4 groups: (1) control;(2) high iodine;(3) poly (I:C);(4) high iodine+poly (I:C). Nine weeks after administration, mice were sacrificed. The following parameters were determined: body weight, thyroid weight and anatomic form. Thyroid hormone (T_4) in serum was measured by radioimmunoassay, the thyroid morphology was observed through HE staining, apoptosis was detected by TUNEL, the mRNA expression levels of TRAIL, TRAIL-sR1, ICAM-1 and CXCL10 were determined by the method of real time RT-PCR. RESULTS: Compared to control group and poly (I:C) group, the thyroid absolute weight and relative weight in high iodine group were increased (P<0.01), the level of total T_4 in serum was decreased (P<0.05), inflammation and apoptosis were obviously observed, the mRNA expressions of TRAIL, TRAIL-sR1, CXCL10 and ICAM-1 were upregulated (P<0.05). Compared to high iodine group, thyroid absolute weight and relative weight in high iodine+poly (I:C) group were further increased, the level of total T_4 in serum was further decreased (P<0.05), the ratio of inflammatory degree Ⅳ increased to 50.0%, the numbers of apoptosis cells were further enhanced, the mRNA expressions of TRAIL, TRAIL-sR1, ICAM-1 and CXCL10 were further upregulated (P<0.05). Otherwise, the tendency of all parameters in poly (I:C) group was similar to that in control group (P>0.05). CONCLUSION: Poly (I:C) aggravates chronic lymphocytic thyroiditis induced by excess of iodine associated with increase in infiltration of lymphocytes and induction of apoptosis.
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AIM: To obtain high level expression of recombinant human truncated osteoprotegerin (TOPG) with higher bioactivity in CHO-DHFR~-cells. METHODS: The recombinant vector pcDNA3.1/DHFR-TOPG was constructed and transfected into CHO-DHFR~- Cells by the directions of LipofectAMINE~(TM)2000 for stable expression. The stable expression cell strains were screened by selective medium IMDM with 50 mL/L FCS, then serially passed in methotraxate (MTX) for gene amplification. The expression were analyzed by ELISA and RT-PCR. At last, the bioactivity analysis was performed in vitro. RESULTS: The expression level of recombinant truncated human OPG was up to 6 mg/L·72 h, and it had significant suppression effect on the formation of OLC(P<0.05). CONCLUSION: Recombinant truncated human OPG has high expression and bioactivity. The results make it possible for further studying and clinical implying of OPG.
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Objective To observe the pathological characteristics of thyroiditis induced by iodine excess and thyroglobulin (Tg) immunization and to explore the mechanism of thyroiditis induced by iodine excess. Methods NOD mice were used for intaking 0.05% Nal water and(or) Tg immunization. Morphologic change in thyroid and apoptosis were observed. The levels of serum TT4, TSH, thyroglobulin antibody (TgAb) and thyroid peroxidase antibody (TPOAb) were measured. Responding to Tg, lymphocytic proliferation of lymph node and spleen, interleukin-4(IL-4)and γ-interferon(IFN-γ) levels in culture medium of splenocytes were detected. Real-time PCR Was used to detect mRNA expressions of IL-4, IFN-γ, chemokine ligand 10 (CXCL10) and intercellular adhesion molecular-1(ICAM-1) in thyroid. Results Distended thyroid follicles,colloid accumulation, intense lymphocytic infiltration and disorganization were seen in thyroid of iodine excess group, along with increased apoptosis of thyroid cells(34.66～ 2.78 vs 5.11±0.62 ,P<0.01). The levels of TT4 were lowered while TSH raised ,but no production of thyroid-specific autoantibodies was revealed. Lymph node and spleen cells showed positive respornse under stimulation of Tg. The level of IFN-γ[(1. 272±0.049 vs 1. 139±0. 025)ng/L,P<0. 01] was raised in culture medium of splenocytes but not IL-4. The expression of IFN-γ, CXCLI0 and ICAM-1 mRNA were increased in thyroid. But in Tg group, some lymphocytes were scattered in thyroid, autoantibodies emerged ,and the level of IL-4 was increased in cuhure medium of splenocytes[(18. 508±0. 113 vs 13. 368±0. 016)ng/L, P<0. 01]. ledine excess combined with Tg enhanced these inflammatory reaction. Conclusion Iodine excess induced thyroiditis in NOD mice. The process seems to be Th1 response dominant organ-specific autoimmune diseases. Iodine excess and Tg immunizatiou play a synergistic role in inducing experimental autoimmune thyroiditis.
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BACKGROUND: Triiodothyronine (T3) is an important regulation factor at the critical period of brain development. It maybe control the successive differentiation during the development of central nervous system (CNS).OBJECTIVE: To monitor the differentiation of neural stem cells (NSCs) induced by T3 and the thyroid hormone receptor (TR) mRNA expression changes.DESIGN: Open experiment.SETTING: Department of Pathology, Tianjin Medical College of Chinese People's Armed Police Force; Institute of Endocrinology of Tianjin Medical University.MATERIALS: This study was carried out in the Tianjin Medical University between January 2003 and March 2005.Ten-to-twelve-week-old aborted fetuses were obtained from the General Hospital of Tianjin Medical University with the approval of the local ethical committee. Informed consents were obtained from the mothers and their relatives.METHODS: ①Under the aseptic condition, the bilateral cortex of human fetal brain was removed and dissociated by brief mechanical trituration in D-Hanks. Then, 20 μg/L bFGF and 30 nmol/L T3 were used to induce the proliferation of NSCs and inoculated to poly-L-lysine-coated 24-well plate and 25 mL culture flask for routine culture at 1 ×109 L-1. The culture medium was DMEM/F12 serum-free complemented with N2. Half of the culture medium was changed every 48 hours.Seven days later, bFGF was discarded, only T3 was used for induction and differentiation. ② At 1, 2 and 3 weeks of culture, cells were collected, and RT-PCR was semiquantitatively used to detect TR mRNA expression changes at different stages of differentiation of NSCs. Isoforms were identified by immuocytochemistry.MAIN OUTCOME MEASURES: ①Cellular morphology observation and isoforms identification before and after differentiation of NSCs induced by T3. ② TR mRNA expression changes during the differentiation of NSCs.RESULTS: ①The hNSCs were round and had a smooth surface and gradually gathered to neurospheres. The proliferative hNSCs were nestin-positive and incorporated BrdU. When NSCs were induced by T3 for one week, most of the cells took on monopole or double poles, and had long and thin processes. The differentiated cells were neurofilament protein (NFP)-positive, galactocerebroside (GC)-positive or glial fibrillary acidic protein (GFAP)-positive. When NSCs were induced by T3 for three weeks, most of the cells were big, with unclear cell membrane, round nucleus, many thick processes which had many branches. The spider-like cells were scattered, and 80% of the cells were myelin basic protein-positive. ② TRα1 mRNA expression level was the highest before inducing NSCs. With the induction of T3, the expression level was decreased gradually, and was the lowest at 2 weeks, and then was rebounded gradually, but the final level was still lower than that of NSC (F =32.49, P =0.008). The tendency of TRα2 mRNA expression alteration was identical with that of TRα1 mRNA. TRβ1 mRNA expression level was the lowest in NSC, was increased gradually with the induction of T3 and attained the highest level at 2 weeks of induction of T3. Furthermore, the expression level of TRβ1 mRNA was also higher than that of TRα1 at the same time (t =15.64,P =0.001), and it reached the lowest level at 3 weeks of the induction. TRα3 expression level was firstly decreased after the differentiation induced by T3, and was close to the expression level of NSC at 2 weeks of induction (F =51.94, P =0.378), then was decreased to lower lever.CONCLUSION: T3 can induce NSC to differentiate into neurons, oligodendrocyte and astrocytes. TR mRNAs are expressed in different time intervals during the differentiation of NSCs.
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Objective To observe the variations of thyroid hormone receptors(RTs) mRNA experession during the human brain devlopment. Methods We investigated the ontogeny of TR isoforms in the first and second trimester human fetal different brain areas by semi-quantitative reverse transcriptase-polymerase chain reaction analysis. When we amplified the TR? 2 by PCR, the other sequence was amplified at the same time, it is about 100pb less than the RT? 2, so we cloned it into pGEM-T easy vector to determine its sequence. Results In the first and second trimester human fatal brain, RTs mRNAs were detected in cerebrum, cerebellum, brain, stem, hippocampus, spinal cord, thalamus. TRs mRNAs were relatively higher in cerebrum, cerebellum, hippocampus. In the first trimester human fatal brain, the TR? isoforms mRNAs were higher than TR? 1, In the second trimester human fatal brain, the TR? 2 and TR? 1 were higher than TR? 1. An additional truncated species was detected with the TR? 2 primer set. We submitted its sequencing results to Genbank, comparing it with TR? 2 by BLAST software, the results showed that it is identical to TR? 2 with the exception that it is missing 42 amino acids at 371-412 of TR? 2 sequence, so it is the human TR? 3. At the same time we acquired the Genbank accession number AF522368. Conclusion The spatial and temporal expressions of TR isoforms mRNAs exist in CNS development. We firstly assure the different sequence between human TR?2 and TR?3.
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AIM:To investigate the effects of poly (I:C) as virus mimics on iodine excess-induced chronic lymphocytic thyroiditis in NOD mouse. METHODS:Female,32 NOD mice were randomly divided into 4 groups:(1) control; (2) high iodine; (3) poly (I:C); (4) high iodine+poly (I:C). Nine weeks after administration,mice were sacrificed. The following parameters were determined:body weight,thyroid weight and anatomic form. Thyroid hormone (T4) in serum was measured by radioimmunoassay,the thyroid morphology was observed through HE staining,apoptosis was detected by TUNEL,the mRNA expression levels of TRAIL,TRAIL-sR1,ICAM-1 and CXCL10 were determined by the method of real time RT-PCR. RESULTS:Compared to control group and poly (I:C) group,the thyroid absolute weight and relative weight in high iodine group were increased (P0.05). CONCLUSION:Poly (I:C) aggravates chronic lymphocytic thyroiditis induced by excess of iodine associated with increase in infiltration of lymphocytes and induction of apoptosis.