稳定表达全长人促甲状腺激素受体细胞株构建及功能评价

Stable expression of the human TSH receptor in CHO ceils and the functional assessment

目的建立能稳定表达全长人促甲状腺激素受体的细胞株并对其进行功能鉴定。方法利用PCR方法钓取全长人促甲状腺激素受体（hTSHR）基因，纯化后交换进入线性化GV208载体，构建出重组载体，载体转化后在辅助包装原件载体质粒的帮助下包装成慢病毒颗粒，感染中国仓鼠卵巢细胞（CHO），单克隆培养后筛选出稳定表达hTSHR的细胞，用qPCR检测其mRNA表达情况。用牛TSH（bTSH）或Graves病患者血清刺激细胞，检测上清cAMP浓度变化来鉴定构建的细胞株功能。结果基因检测证明阳性转化子目的基因序列正确。与对照组或空白组细胞相比，构建的实验组细胞株表达的hTSHRmRNA升高，在受到bTSH刺激时，随着bTSH浓度增加，cAMP明显增高，初发Graves病患者血清刺激细胞时，与对照组血清相比cAMP明显增高。结论构建的CHO细胞株能稳定表达全长人促甲状腺激素受体，并且该细胞株功能良好，可用于后续研究。

Objective To establish and assess a CHO （Chinese Hamster Ovary ） cell line that stably expressed the human TSH receptor. Methods The gene of human TSH receptor was amplified by PCR from the recombinant plasmid that bad been constructed previously, and then the target gene was linked to linearized vector GV208. Afterwards, with the reconstructed vector and helper vectors, the target gene was packaged into the lentivirus. The recombinant lentivirus infected the CHO cells and integrated the gene into the chromatin. The established CHO cell line was isolated and cultured and its function was assessed. Results The result of gene test identified the correct sequence of DNA of the transformant. The expression of mRNA in CHO cells transfected with the human TSHR was obviously higher than the cells in control or blank group. Within the range of certain density, the more bovine TSH was used to stimulate the established CHO, the more cAMP would be produced. When stimulated with serum of patients of Graves＇ disease, the cell line also significantly produced more cAMP. Conclusion The established CHO cell line transfected with the human TSHR can stably express the human TSH receptor and response to the stimulation of ligand of TSH receptor.