【作者】 李靖；

【导师】 刘皓； 李兰英；

【作者基本信息】 天津医科大学 ， 人体解剖与组织胚胎学， 2010， 硕士

【摘要】 目的已知除了垂体,免疫系统也能产生TSH。最近科学家发现一种骨髓造血细胞可以表达TSHβ剪切体并可迁移到甲状腺组织表达该物质。SHβ剪切体在垂体、骨髓和甲状腺都有表达,但脾作为机体重要的免疫器官,是否表达这一剪切体尚未证实,且该剪切体的病理生理学意义尚且不清。因此我们要确认脾组织是否表达TSHβ剪切体；通过检测TSHβ剪切体在碘过量和甲状腺球蛋白诱发的自身免疫性甲状腺炎中小鼠不同器官表达的变化,探讨发生甲状腺炎时,TSHβ剪切体对甲状腺功能发挥的作用,初步了解TSHβ剪切体与甲状腺炎发病的关系。以便更深入研究TSHP剪切体在下丘脑-垂体-甲状腺轴以及在免疫-内分泌相互作用的中的具体作用和机制。方法本实验首先选择20只雌性自身免疫性疾病易感的NOD小鼠,7-8周龄,随机分为4组,每组5只。分别为：对照组(C)：饮用去离子水；碘过量组(HI)：饮用0.05%NaI水；TG组(TG)：饮用去离子水,8周龄时给予每只鼠0.1mgTG皮下免疫,11周龄、15周龄加强免疫两次；碘过量+TG组(HI+TG)：饮0.05%NaI水,TG免疫同上。取动物全血,采用RIA法和ELISA法分别检测小鼠血清中甲状腺激素和促甲状腺素水平。然后对垂体和脾组织进行TSHβ免疫荧光染色,观察TSHβ阳性细胞在脾的分布。使用PCR技术对TSHβ剪切体在NOD小鼠垂体、骨髓、脾与甲状腺组织中表达进行检测,并对脾组织该基因的扩增产物进行测序。用SYBR GREEN实时荧光定量PCR法分析不同处理因素下TSHP剪切体基因mRNA表达。结果TG组与C组比较血清T4水平和TSH水平都增加。C组T4水平为(36.43±3.87) nmol/L, TSH水平为(5.55±0.28) ng/mL。TG组血清T4水平为(69.70±16.45) nmol/L (P<0.05)和TSH水平为(8.44±0.71) ng/mL (P<0.01)。HI组和HI+TG组TT4水平较C组无统计学差异,而TSH水平较C组升高,分别为(6.98±0.27) ng/mL (P<0.05)和(9.12±0.36) ng/mL (P<0.01)。脾组织TSHβ阳性细胞主要位于边缘区,生发中心有少量阳性细胞,但不能确认是天然型TSHβ还是TSHβ剪接体；垂体可见大量TSHβ阳性细胞。RT-PCR技术显示骨髓、垂体、甲状腺和脾都表达TSHβ剪接体,但是除垂体外其他组织都不表达自然型TSHβ。TG组骨髓TSHβ剪接体表达(0.026±0.010)较C组(0.014±0.007)增加(P<0.05),垂体和甲状腺TSHβ剪接体表达与C组比无明显差异,垂体自然型TSHβ表达与C组比也无明显差异。HI组骨髓TSHβ剪接体表达与C组比较无差异；HI组垂体TSHβ剪接体表达与C组比较明显增加(P<0.01),HI组为(0.038±0.010),C组为(0.015±0.007)；HI组甲状腺TSHβ剪切体表达较C组增加(P<0.05),HI组为(0.036±0.007),C组为(0.017±0.003)；垂体自然型TSHβ表达(0.013±0.003)与C组(0.006±0.000)比增加(P<0.01)。HI+TG组只有垂体自然型TSHβ(0.015±0.004)与C组比较,表达有增加(P<0.01)。各处理组脾组织TSHβ剪切体表达未做出结果。结论NOD小鼠脾脏作为机体重要的免疫器官也可以表达TSHβ剪切体。TG免疫可使血中TSH水平升高,与源于骨髓细胞分泌的TSHβ剪切体表达有关,骨髓TSHβ剪切体的表达可能不受血中T4的负反馈调节；碘过量使T4水平下降,垂体中天然型TSHβmRNA的表达升高,其变化符合HPT轴的反馈学说,而垂体TSHβ剪切体表达的调控还有待于进一步研究验证；碘过量组与TG免疫组甲状腺TSHβ剪切体表达不一致,提示与碘过量诱发的EAT是以细胞免疫为主的炎症,所造成的炎症反应和组织破坏程度较TG免疫更为严重,导致向甲状腺迁移的分泌TSHβ剪切体的骨髓细胞增加有关。更多还原

【Abstract】 Objective:As we all know in addition to pituitary, immune system can produce TSH. Scientists recently found a set of resident intrathyroidal bone marrow-derived hematopoietic cells which can express a novel TSHP splice variant. The TSHP splice variant gene was expressed in the pituitary, bone marrow and the thyroid. Spleen is an important immune organ which expresses TSHβ, we need to confirm it is native TSHP or TSHβsplice variant. We detected the expression of TSHP splice variant in different organs on autoimmune thyroiditis induced by iodine excess and thyroglobulin in NOD mice, to explore the function of TSHP splice variant in infection-or inflammatory-related thyroid disorders.Methods:Twenty Non-obese diabetic mice,7-8 weeks old, female, were randomly divided randomly into four groups:control group (C):drank deionized water; iodine excess group (HI):drank 0.05% NaI water; TG group (TG):drank deionized water, immunized with 0.1mg TG to each mouse by subcutaneously injection at 8 weeks old and enhanced at 11 and 15 weeks old; iodine excess+TG group (HI+TG):drank 0.05% NaI water, TG immunization was the same as TG group. Then collected blood samples, RIA and ELISA were used respectively to detect T4 and TSH level in serum.For observing distribution of TSHP positive cells, TSHβimmunofluorescence staining was used on pituitary, and spleen. The expression of TSHP splice variant mRNA were detected by real time PCR in different organs and the amplified gene of spleen was sequenced. SYBR Green real-time PCR was used to detect the levels of TSHP splice variant genes in different organs.Results:TG group had a higher level of T4 and TSH compared to C group. The level of T4 and TSH in C group were separately (36.43±3.87) nmol/L and (5.55±0.28) ng/mL, while the level of T4 and TSH in TG group were(69.70±16.45)nmol/L(P<0.05) and (8.44±0.71)ng/mL (P<0.01).The level of T4 in HI group and HI+TG group had no significant different compared to C group. But HI group and HI+TG group had higher level of TSH than C group, were (6.98±0.27)ng/mL (P<0.05) and (9.12±0.36)ng/mL(P<0.01) separately.In spleen, majority of TSHβ+ cells were localized in the marginal zones, few of which localized in germinal center. In pituitary, there were large number of TSHβ+ cells.RT-PCR showed that TSHP splice variant was not only expressed in the pituitary, bone marrow and thyroid but also in the spleen. Native TSHP was only expressed in pituitary. In TG group, expression of TSHP splice variant in bone marrow (0.026±0.010) (P<0.05) increased compared to C group (0.014±0.007), while pituitary and thyroid had no higher expression of TSHβsplice variant than C group. Native TSHβin pituitary had no significant different either. In HI group, expression of TSHP splice variant in bone marrow did not changed significantly, but expression of TSHP splice variant in pituitary(0.038±0.010)(P<0.01) and thyroid(0.036±0.007)(P<0.05) were higher than C group(0.017±0.003). Native TSHP in pituitary was higher (0.013±0.003) (P<0.01). In HI+TG group only the expression of native TSHP in pituitary increased (0.015±0.004) (P<0.01). TSHp splice variant expression in spleen had no significant result.Conclusions:Spleen as an important immune organ in NOD mice can express TSHP splice variant. Increased level of TSH in TG group had something to do with the expression of bone marrow TSHP splice variant. Its production and secretion may not be subject to the negative feedback regulation of circulating T4. In TG group, pituitary expression of native TSHβmRNA meet the HPT axis feedback theory, but the regulation of expression of TSHβsplice variant in pituitary needs further research. Expression of TSHP splice variant of thyroid in HI group and TG group was different, suggesting that excess iodine-induced EAT is a cellular immune-based inflammation, the inflammatory response and tissue damage are more serious than the TG immunity, leading to more bone marrow cells that express TSHP splice variant migrate to the thyroid.更多还原