| Objective:In this experiment, we treated the different strain mice with drinking iodine excess water, Tg immunization and both. Then observed the thyroid morphology and function change, cytokines secretion by spleen lymphocytes, if and where inflammation and apoptosis related receptor/ligand emerged in thyroid, the level of inflammation and apoptosis related gene expressed in thytoid of both strain mice. Thus to reveal the distinct influence of environmental factors on inducing individuals with different genetic background to develop autoimmune thyroiditis (AT), investigate the mechanisms of lymphocytic infiltration in thyroid and epithelial cell apoptosis in AT and to provide experimental foundation for reveal etiological factor and pathogenesy of human autoimmune thyroiditis.Methods:Female,7-8 weeks old NOD and BALB/c mice were used and randomly devided into four groups:control group (C):drank deionized water, Tg group (Tg): drank deionized water, immunized with 0.1 mg Tg to each mouse by subcutaneously injection at 8 weeks old and enhanced at 11 and 15 weeks old, iodine excess group (HI):received 0.05% NaI water, iodine excess+Tg group (HI+Tg):received 0.05% NaI water, Tg immunization was same as Tg group. At 16 weeks old, we weighed mice body weight, collected blood samples, observed the thyroid morphous and measured the weight of the thyroid after mice were sacrificed. HE staining was employed to observe the histopathology of thyroid, TUNEL was used to detect the apoptosis of thyroid follicle epithelial cells, RIA and ELISA were used respectively to detect TT4,TgAb,TPOAb and TSH level in serum. MTT method for lymphocytic proliferation, IL-4, IFN-y, IL-10, IL-12 levels in splenocyte culture supernatants were assayed by ELISA. Immunofluorescence staining to observe if and where CD3, CD22, CD31, Podoplanin, CCL21, CCR7, TRAIL and TRAIL-R2 (DR5) emerged in thyroid. SYBR Green real-time PCR was used to detect the levels of inflammation related genes (CCL21, ICAM-1 and CXCL10) and apoptosis related genes (TRAIL and TRAIL-sRl (DR4)) expression.Results:1. Morphology change of thyroid: for the mice of two strain, enlargement of the thyroid was observed in the HI and HI+Tg group than C group, the absolute and relative weight were increased (P<0.01), and enlarged follicular lumen with colloid accumulation was observed. For NOD mice thyroid of HI and HI+Tg group, focus lymphocytic infiltration was seen, partly fibrosis was also seen in HI+Tg group.2. Hormore and autoantibodies leve in serum: for two strain mice, the HI group had a lower level of TT4 and a higher level of TSH compared with C group. But the level of TT4 and TSH in Tg and HI+Tg group were both higher than their C group (P<0.01). TgAb, TPOAb were producted in two groups performed Tg immunization (P<0.01).3. Lymphocytic proliferation and cytokines level in splenocyte culture supernatants: after stimulated with Tg in vitro, compared with C group, lymphocytic proliferation increased in each experiment group of two strain mice (P<0.01) except HI group of BALB/c mice. Cytokines level:for NOD mice, cytokines level were all elevated in each experiment group to C group (P<0.05 or 0.01) except IL-4 level in HI group. For BALB/c mice, IL-4, IL-10 and IFN-γlevel elevated in Tg and HI+Tg group (P<0.01), no change in HI group, IL-12 level decreased in all experiment groups (P<0.01).4. Apoptosis of thyroid follicle cells:C group thyroid of two strain mice have few weakly positive cells. For NOD mice, large amount of positive cells were observed in HI and HI+Tg groups, but for Tg group and experiment groups of BALB/c mice, postive cells were only a little more than C groups.5. TRAIL and its receptor expression in thyroid:Immunofluorescecce staining: In C group thyroid of NOD mice, TRAIL and DR5 were negative. Scattered TRAIL and DR5 positive expression in Tg group, their expression increased in HI group, and more in HI+Tg group. TRAIL was expressed on thyroid follicle epithelial cells and infiltrating inflammatory cells, the expression intension on inflammatory cells is stronger than on thyrocytes, DR5 located on thyroid follicular epithelial cells. For BALB/c mice, small amout of TRAIL expressed on thyrocytes or inflammatory cells in each experiment group, weaker than NOD mice. No DR5 expression in BALB/c mice.Genetic expression:For NOD mice thyroid, TRAIL expression increased in all experiment groups (P<0.01), DR4 increased in HI and HI+Tg groups except Tg group (P<0.01). In BALB/c mice, TRAIL in Tg and HI+Tg group (P<0.05 and 0.01) and DR4 in HI+Tg group increased (P<0.01).6. Inflammation related gene expression in thyroid:Immunofluorescecce staining:(1) No CD3 positive cell in C group. For NOD mice thyroid, scattered CD3 positive cells in Tg group, large amount of CD3 positive cells were seen in HI and HI+Tg group and HI+Tg group has a bigger area and fibration was emerged. BALB/c mice, CD3 positive cells were very few in Tg and HI groups, a little more positive cells in HI+Tg group. No CD22 positive cells in BALB/c mice thyroid. NOD mice, two or three CD22 positive cells in Tg group, no CD22 positive cell in HI group, few CD22 positive cells in HI+Tg group. (2) CCL21 and CCR7 were negative in C group thyroid in NOD mice. CCR7 positive cells were scattered in Tg group but no CCL21 expression. In HI and HI+Tg group, we can see CCL21 strong positive expression in inflammation area and also has CCR7 positive cells there. The expression of CCL21 located in stroma and lumens like. BALB/c mice thyroid did not have CCL21 and CCR7 expression.Genetic expression: ICAM1, CXCL10, CCL21 mRNA expression were all increased in three experiment groups compared with C group in NOD mice (P<0.01). For BALB/c mice, ICAM1, CXCL10 mRNA expression increased in HI and HI+Tg group (P<0.05 and 0.01), CCL21 expression did not change in BALB/c mice thyroid.Conclusions:1. Iodine excess can induce EAT in sensitive mice but not in non-sensitive mice, indicating that intake excessive iodine is only an important environmental factor; the genetic background determines the onset of HT.2. Both iodine excess and Tg immunization can induce sensitive mice EAT, but by different ways. Tg as an autoantigen can stimulate the autoimmune response, iodine excess is mainly by thyrocytes damage to induce EAT. Tg immunization combine iodine excess aggravate inflammation in EAT.3. Lymphocytes of two strain mice had different reaction to Tg stimulation. Cell proliferation reaction in BALB/ c mice was stronger than NOD mice. In NOD mice experiment groups, both Th1 and Th2 cytokines elevated under their competitive and inhibitive interactions though not significantly elevated. But for BALB/c mice, the basic level of Th1 and Th2 cytokines were higher than NOD mice and Th2 reaction was stronger than Th1 reaction.4. TRAIL binding its receptors DR4 or DR5 can promote thyroid follicular epithelial cells apoptosis in AT. TRAIL induced apoptosis was positive correlation with the extent of inflammatory cells infiltration, but determined by the level of death receptors expression, ligand and receptor not a single one can be omitted.5. Chemokine CCL21 and its receptor CCR7 are important in lymphocytic infiltration in AT. When thyroid suffer inflammation inducing factors, CCL21 with CCR7 can recruitment lymphocytes to thyroid from circulatory system.6. ICAM-1 and CXCL10 expression increased in thyroiditis, indicating that they have adhesive attraction and chemotaxis during lymphocytic infiltration.
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