反义肽核酸对促甲状腺激素受体mRNA表达的影响

目的探讨反义肽核酸（asPNA）对大鼠甲状腺细胞的细胞膜表面促甲状腺激素受体（TSHR）表达的影响。方法设计两种与TSHRmRNA不同片段互补的asPNA，分别为核定位信号-asPNA1（NLS—asPNA1）和NLS．asPNA2，另外合成一段非相关的干扰肽核酸（NLS—scrPNA）序列，用荧光显微镜观察NLS—asPNA能否进入细胞，采用噻唑蓝法检测asPNA对细胞是否具有毒性作用，然后用实时定量RT—PCR检测NLS—asPNA对TSHRmRNA表达的影响。结果荧光显微镜观察发现，asPNA可以进入到细胞中去。噻唑蓝法检测发现asPNA对细胞没有毒性作用。实时定量RT—PCR结果显示，NLS—asPNA1和NLS—asPNA2可以抑制TSHRmRNA表达，表现为随着NLS—asPNA与细胞培养时间的延长，TSHRcDNA的拷贝数逐渐下降，且呈现时间依赖性，而NLS．scrPNA对TSHRtuRNA的表达没有明显的影响。结论NLS—asPNA可以进入到细胞中去，并且能够下调TSHRmRNA的表达。

The effects of antisense peptide nucleic acid on the expression of thyroid stimulating hormone receptor mRNA

Objective To study the effects of antisense peptide nucleic aeid（asPNA） on thyroid stimulating hormone reeeptor（TSHR） of Fisher rat thyroid cells membrane. Methods Two kinds of asPNA which could hybridized to the TSHR mRNA named as nuclear localization signal-asPNAl（NLS-asPNA1） and NLS-asPNA2 were designed, and a control PNA with a scramble sequence named as NLS-scrPNA was syn- thetized. The eellular uptake of peptide nucleic acids were analyzed by fluorescence microscopy, and the toxic effect of asPNA to Fisher rat thyroid cells was evaluated by MTT assay. The effect of NLS-asPNA on the expression of TSHR tuRN A was detected by realtime quantitative RT-PCR. Results Fluorescence microscopy indicated that asPNA were taken up by the cells. The results of MTT assay showed that all the asPNA had no toxic effect on the cells. The results of realtime quantitative RT-PCR showed that NLS-asPNA1 and NLS-asPNA2 could inhibit tile expression of TSHR mRNA, the copies of TSHR eDNA decreased gradually along with the prolongation of culture time. But NLS-serPNA had no significant effect on the expression of TSHR mRNA. Conclusion NLS-asPNA could be taken up by the ceils and down-regulate the expression of TSHR mRNA.