转染中国仓鼠卵巢细胞人TSH受体的功能评价

目的研究转染中国仓鼠卵巢(CHO)细胞人促甲状腺激素(hTSH)受体(hTSHR)的功能。方法抽提已转染hTSHR的CHO细胞基因组DNA,PCR扩增目的条带,并对产物进行序列测定。采用受体的放射配体结合分析法,以固定量125I的牛促甲状腺激素(125I-bTSH)和浓度递增的未标记bTSH与hTSHR发生竞争结合反应分析受体的基本功能;通过TSH刺激试验测定细胞环磷酸腺苷(cAMP)含量,评价hTSHR的生物学活性。结果PCR扩增片段长度与预期相符,且序列完全正确。竞争结合反应表明,随着bTSH浓度的递增,125I-bTSH与hTSHR的结合量逐渐下降;TSH刺激试验显示,随着bTSH浓度的递增,细胞cAMP含量亦逐渐升高,当bTSH浓度为10 mIU/mL时,cAMP水平达到峰值。结论cDNA序列整合在CHO细胞基因组内并在细胞膜上表达的hTSHR,具有受体的基本功能特性和良好的生物学活性。该实验为自身免疫性甲状腺疾病患者血清甲状腺刺激性抗体(TSAb)和阻断性抗体(TSBAb)的检测奠定了基础。

Functional study of human TSH receptor transfected into Chinese hamster ovary cells

Objective To study the function of human thyroid stimulating hormone(hTSH) receptor(hTSHR) transfected into Chinese hamster ovary(CHO) cells. Methods Genomic cDNA was prepared from CHO cells expressing hTSHR(CHO-hTSHR).The hTSHR cDNA sequences integrated in genome of CHO cells was demonstrated via PCR amplifying and DNA sequencing.The basic function of the recombinant hTSHR was estimated by radioligand binding assay.The bioactivity of the receptor was estimated by measuring cAMP production in response to bovine TSH(bTSH) stimulation. ResultsThe length of PCR product was consistent with expectation,and the sequence was identical with that of hTSHR cDNA in GenBank.In binding assay,with the increasing concentration of unlabled bTSH,the binding of labeled bTSH to receptor decreased correspondingly.In TSH stimulating assay,with the increasing concentration of bTSH,cAMP production increased gradually until the concentration of bTSH at 10 mIU/mL. Conclusion hTSHR cDNA can integrate into genome of subculture CHO-hTSHR cells and the recombinant hTSHR expressing on plasma membrane has favourable bioactivity.CHO-hTSHR cells are suitable for detecting serum thyroid-stimulating antibody(TSAb) and thyroid-stimulating blocking antibody(TSBAb) activity in patients with autoimmune thyroid disease.