环境雌激素多氯联苯（PCBs）影响甲状腺干细胞增殖与分化的研究

We have proved that polychlorinated biphenyls(PCBs) might interfere the thyroid function and thyroid homeostasis by inducing apotosis of primary thyrocytes. In vivo, PCBs affect thyroid function via the induction of autoimmunity. However, the incidence of thyroid tumor is increasing in the population exposing to PCBs. The mechanism has not been fully understood. Recent researches of thyroid stem cells have opened a new pathway for understanding the physiological mechanisms controlling basic biological processes as well as disease mechanisms. The multipotentiality and self-renewal ability of the stem cells are controlled by stem cell niche providing a microenvironment, composed of cellular structures or extracellular matrix in undifferentiated stem cells. Our previous work demonstrated that estrogen has the potential to stimulate the proliferation and inhibit the differentiation of thyroid stem cells.PCBs, refered to as environmental estrogens, has been demonstrated to mimic the estrogenic effect. In view of that, the question was addressed if PCBs alters the functions of adult thyroid stem/progenitor cells during the proliferation or differentiation process, and induces outgrowth of stem cells and aberrant differentiation to transform thyroid nodules. In the current study, we have investigated: 1) the potential of PCBs to promote the proliferation of human thyroid stem/progenitor cells; 2) the influence of PCBs on TSH-induced differentiation of adult thyroid stem and progenitor cells into thyrocytes; 3) determination of gene profiles after PCBs stimulation compared to normal thyrocytes and cancer cells ; 4) tumor formation after transplantation of PCBs cultured thyroid stem cells into experimental nude mices.

本实验组在前期研究中发现，多氯联苯（PCBs）作为一类环境雌激素，能引起原代甲状腺细胞凋亡，和小鼠体内慢性甲状腺炎标志物过氧化物酶抗体（TPOAb）增高、甲状腺素水平下降。而流行病学研究却发现，PCBs污染人群甲状腺肿瘤患病率明显增高。目前，对此尚无合理解释，近年来倍受关注的甲状腺肿瘤干细胞学说为解释这一现象提供新的途径。我们的前期研究发现，甲状腺干细胞可在生长因子刺激和悬浮无血清培养下维持未分化状态，雌激素明显影响促进其增殖，并降低其分化。因此，申请人认为存在于污染环境且具有类雌激素作用的环境雌激素多氯联苯（PCBs）亦能影响甲状腺干细胞的增殖与分化，进而参与甲状腺肿瘤的形成。该研究应用细胞培养等基础研究方法，探讨PCBs是否参与影响甲状腺干细胞的增殖与分化，并观察ER受体及受体后机制在其中扮演的角色，最后在体内实验观察PCBs是否导致甲状腺干细胞异常分化和肿瘤形成。

暴露于多氯联苯（PCBs）的人群甲状腺肿瘤患病率增加。体内体外实验均表明，PCBs可通过引起甲状腺细胞凋亡继而影响甲状腺功能。可引起甲状腺细胞凋亡的PCBs与甲状腺结节和肿瘤形成之间尚无合理解释。在甲状腺内成功鉴定的甲状腺干细胞可能有助于阐释该科学问题。雌激素可促进甲状腺干细胞增殖，并降低其分化。进一步探明具有类雌激素效应的PCBs对甲状腺干细胞的作用及其机制有助于明确环境污染物PCBs与甲状腺肿瘤形成之间的关内在联系。本研究建立人甲状腺干细胞的培养体系，通过体外实验观察PCBs对甲状腺干细胞增殖和分化的影响及雌激素受体在其中发挥的作用，并通过体内实验，证实PCBs刺激后的干细胞是否形成肿瘤。本研究成功建立国内甲状腺干细胞培养体系，甲状腺原代细胞通过第一代悬浮培养后5-7天，形成“甲状腺干细胞球（thyrosphere）”。进一步鉴定显示，干细胞球可稳定表达OCT4等干细胞标志物，而不表达TSHR、TPO、NIS等成熟甲状腺细胞标志物，符合甲状腺干细胞特征。进一步鉴定显示，甲状腺干细胞球的雌激素受体α显著高于成熟甲状腺细胞（8.85±0.81 vs 1.10±0.35, P＜0.001），而雌激素受体β则无差异。通过不同浓度和PCBs、PCB118、PCB126、PCB153对甲状腺干细胞球进行细胞活力检测时，发现低浓度的PCBs及其单组份对甲状腺干细胞无明显促增殖效应，而浓度增高时其可形成结晶。干细胞球形成实验表明，PCBs及其单体未能维持干细胞球形成。而另一类环境雌激素双酚A（BPA）在20 μM时可显著刺激甲状腺干细胞增殖，并维持干细胞球形成。另外，BPA可影响人甲状腺干细胞的分化，并降低钠碘共同转运子（NIS）的表达。本研究证实人正常甲状腺组织中存在一类具有自我更新的成体干细胞，可在生长因子、环境雌激素的作用下形成自我克隆形成干细胞球。不同种类的环境雌激素对甲状腺干细胞促增殖效应可能存在较大差别。这一结果提示环境污染可能与体内甲状腺结节或肿瘤形成具有一定内在联系。