化痰散结法对Graves病小鼠模型甲状腺肿大与激素合成下游事件的影响

Graves' Disease (GD) is characterized by goiter and thyroid hormone overproduction,and Thyrotropin receptor antibody (TRAb) is the key produt of hyperthyroidism in Graves' Disease.Circulating iodide is actively transported into the thyrocyte by the Na+/I-Symporter (NIS).Iodide is catalyzed by thyroid peroxidase(TPO).Thyroglobulin(TG) is the storage form of the iodide in the thyroid. The level of TG is depending on the size of thyroid. These are the three key processes of goiter and downstream of hormone synthesis.TRAb can improve the activity / content of the three substances and lead to goiter. GD belongs to "Yingqi" in TCM. Resolving phlegm to dissipate stagnation tenet is the basic therapy.It has been proved that resolving phlegm to dissipate stagnation can inhibit the thyroid cells proliferation, promote their apoptosis and restore the balance of cytokinetics. This study is targeted at non-marine herbs of resolving phlegm to dissipate stagnation, and the mice model of GD immunized by Ad-TSHR289 as carriers. To study the effection of Resolving phlegm to dissipate stagnation to the goiter and downstream of hormone synthesis by the transcription product of mRNA and the translation of protein of NIS, TPO, Tg. It studies the mechanism of resolving phlegm to dissipate stagnation to inhibite proliferation and promote apoptosis, the apoptosis rate of the cells by Annexin-V, PI, end-labeling, the expression of Bcl-2 family and Fas/FasL mRNA .It demonstrates the effection of resolving phlegm to dissipate stagnation to goiter and the ultrastructural organization of thyroid follicular under the electron microscopy.This research effectively deepen and replenish the previous studies.

弥漫性甲状腺肿伴甲亢（GD）以甲状腺肿伴激素增高为特征，关键是产生TRAb。NIS将碘转运至细胞内、TPO使碘活化、Tg是碘在甲状腺的贮存形式，甲状腺大小决定Tg水平高低，三者是甲肿与激素合成下游三个关键环节，TRAb可提高三者活性/含量，产生甲肿。GD属中医"瘿气"，化痰散结法为基本治法，以往体外研究证实化痰散结法可抑制甲状腺细胞增殖、促进凋亡，恢复细胞动力学平衡。本研究以非海生化痰散结药物为对象，以Ad-TSHR289免疫的GD小鼠模型为载体，通过NIS、TPO、Tg的mRNA和蛋白表达，研究化痰散结法对甲肿与激素合成下游关键事件的作用；采用AV /PI和TUNEL染色检测凋亡比率，通过Bcl-2 家族及Fas/FasL的mRNA表达,研究化痰散结法抑制增生和促进凋亡的机制；通过电镜甲状腺滤泡超微结构研究化痰散结法对甲肿和激素合成与分泌的作用。本研究是以往研究的有力深化和补充。

研究背景：弥漫性甲状腺肿伴甲亢（GD）以甲状腺肿伴激素增高为特征，关键是产生TRAb。NIS将碘转运至细胞内、TPO使碘活化、Tg是碘在甲状腺的贮存形式，甲状腺大小决定Tg水平高低，三者是甲肿与激素合成下游三个关键环节，TRAb可提高三者活性/含量，产生甲肿。GD属中医“瘿气”，化痰散结法为基本治法，以往体外研究证实化痰散结法可抑制甲状腺细胞增殖、促进凋亡，恢复细胞动力学平衡。本研究以非海生化痰散结药物为对象，以Ad-TSHR289免疫制备的GD小鼠模型为载体，重点研究化痰散结法对甲状腺激素合成关键环节NIS、TPO、Tg的表达，甲状腺组织肿大形态及超微组织病理变化，甲状腺细胞增殖凋亡的及Treg/IL-17细胞免疫调节机制的影响。.研究内容、结果及关键数据：化痰散结药物干预小鼠与模型组相比：（1）甲状腺内的NISmRNA、TPO mRNA及Tg mRNA显著的减低（P<0.01），经Western-Blot检测后，其甲状腺内Tg的表达低于模型组。（2）小鼠血清T4下降18.73%（P=0.021）；TRAb下降35.47%（P=0.000）。（3）甲状腺肿大减轻，充血改善，滤泡腔内的乳头状凸起减少，滤泡上皮细胞增生及肥大得到改善，高柱状细胞形态逐渐向扁平状恢复，滤泡腔内的胶质增加等。（4）甲状腺组织的PCNA阳性的甲状腺滤泡上皮细胞减少，Fas阳性、FasL阳性的甲状腺滤泡上皮细胞增加，Bax阳性的甲状腺滤泡上皮细胞增加。（5）小鼠脾脏淋巴细胞中CD4+CD25+Foxp3+ Treg细胞的比例上升73.62%；脾脏淋巴细胞中Foxp3 mRNA的表达上升186.67%，IL-17 mRNA的表达下降71.27%。.阐释了化痰散结法能够下调激素合成下游三个关键因子表达，从而改善GD小鼠甲亢状态；其使甲状腺组织肿大得到改善可能是通过抑制细胞增殖核抗原PCNA的表达，同时上调细胞凋亡相关因子Bcl-2、Fas等的表达，从而抑制甲状腺细胞增殖促进细胞凋亡。同时对GD免疫紊乱具有一定的调节作用。此外，通过构建优化腺病毒载体pAd-TSHR289/6xHis，GD成模率由早期的70%升至100%。.科学意义：本项目为中医化痰散结法治疗GD甲状腺肿大的机理提供了现代科学依据，为后续开展免疫学相关研究做出探索，对临床有针对性选择治疗甲状腺肿大的药物具有重要意义。