摘要
目的分离培养小鼠甲状腺细胞,为探讨甲状腺功能调节和疾病发生提供一种体外模型。方法取6～8周龄Balb/c小鼠甲状腺,用胶原酶-中性蛋白酶双酶法分离甲状腺滤泡上皮细胞。用含10%胎牛血清(FCS)的F-12培养基培养,细胞贴壁后FCS降至5%,另外添加促甲状腺激素(TSH)、氢化可的松、转铁蛋白、胰岛素和L-谷氨酰胺。光镜下对不同培养时间的细胞进行形态学观察,并结合免疫细胞化学染色法、RT-PCR法和电化学发光免疫法对培养小鼠甲状腺细胞从形态、甲状腺球蛋白(T_g)、T_g特异抗原蛋白表达、甲状腺过氧化物酶(TPO)、钠碘转运子(NIS)、促甲状腺激素受体(TSH-R)等特异基因mRNA表达和甲状腺激素T_3、T_4分泌功能等方面进行鉴定。结果小鼠甲状腺细胞在体外呈贴壁式生长,具有上皮样细胞特点;Tg染色呈胞浆阳性,并具有特异TPO、Tg、NIS、TSH-R mRNA表达和分泌T_3、T_4的功能,但其表达与分泌量有随培养时间的延长呈递减趋势。结论成功建立小鼠甲状腺细胞原代培养模型,可用于甲状腺功能与疾病研究。
Objective To isolate and culture mouse thyroid cells as a novel in vitro model for studying thyroid pathophysiology. Methods Thyroids were excised from 6 - 8 week-old BALB/c mice and digested with enzymes to obtain thyroid follicular cells which were cultured in F-12 medium supplemented with 5% - 10% FCS and containing thyrotropin （TSH）, insulin, hydrocortisone, transferrin, L-Glutamic acid. Thyroid specific antigen thyroglobulin（Tg） expression was detected by immunohistochemical staining. The expression of thyroid peroxidase （TPO）, thyroglobulin（Tg）, sodium/iodide symporter（NIS）, and TSH-receptor as well as secretion of thyroid hormones by cultured cells were assessed by RT-PCR and electro-chemiluminescence immunoassay （ECLIA）, respectively. Results The cultured cells showed the characteristics of attached epithelial-like cells and strong cytoplasmic staining for Tg. The cells expressed TPO, Tg, NIS and TSH-R at mRNA levels and were able to release T3 and T4 for up to 16 days after seeding, although the expressing and secreting gradually decreased with time. Conclusions Primary cultures of functional mouse thyrocytes have been established, which can be used for the explorations of gene expression, functional regulation and diseases of thyroid.
出处
《中国地方病学杂志》
CAS
CSCD
北大核心
2007年第6期620-623,共4页
Chinese Jouranl of Endemiology
