The Effects Of Thyrotropin Receptor Antisense Peptide Nucleic Acid Treatment On Graves' Disease Animal Model

AIM: To make Graves' disease animal model and to study the effects of TSHR asPNA treatment on Graves' disease animal model.METHODS: 1. Two cDNA sequences encoding hTSHR extracellular domain fragments (AA29-280, AA314-418) were recombined with eukaryotic expression vector pcDNA3.1 (D) /HIS-V5- TOPO. And the recombinant plasmids expressed TSHRa and TSHRc in CHO cells. Then BALB/c mice were immunized with the recombinant plasmids and serum T₄, TRAb, TSAb, TBAb and thyroid TSHR mRNA levels were assayed. Thyroids were subjected to pathological examination when the mice were sacrificed. 2. According to TSHR gene sequence published in Genebank, two asPNAs were designed to investigate their effects on TSHR mRNA level in FRTL cells, which matched with the first ATG and 5' UTR, respectively. Then Graves' disease mice model received i.p. injections of asPNAs after it was proved that asPNAs could inhibit TSHR mRNA level in vitro. To study the effects of asPNAs on Graves' disease, serum T4, TRAb, TSAb and thyroid TSHR mRNA levels are assayed. And thyroids were subjected to pathological examination when the mice were sacrificed.RESULTS:1. Recombinant expression plasmids pcDNA3.1 TOPO/TSHRa and pcDNA3.1TOPO / TSHRc were successfully constructed. Proteins (TSHRa and TSHRc) were expressed in CHO cells transfected by recombinant plasmids, with molecular weight 32.2 kD and 15.9 kD respectively, which were consistent withexpectation. 2. In the mice treated with TSHRa, TSHRc gene, elevated TRAb at week 6 were observed. T4 and TSAb index values were significantly increased at week 10. Through thyroid pathological examination, proliferated thyroid follicular epithelial cells, follicular capacity increase and condensed colloid substance were observed. Inflammatory cells were found occasionally. 3. In the BALB/c mice immunized with KLH-TSHR38- 45 and gTSHR, TRAb, TSAb and T4 increased at week 6 and week 12. In thyroid pathological examination, proliferated thyroid follicular epithelial cells, follicular capacity increase and condensed colloid substance were observed. Lymphocyte infiltration was not seen. The model is proved stable and not mixed with thyroid hypofunction. 4. TSHR NLS-asPNAs reduced TSHR mRNA level, not only in vitro, but also in vivo. TSHR mRNA levels decreased by 55% and 38% in FRTL treated with NLS-asPNAs for 24 h and by 83% and 67% for 48 h, respectively. Similarly, thyroid TSHR mRNA levels (1649±224,1478± 1173 copies) were lower in NLS-asPNAs-treated mice than control (2806±680 copies) (P<0.05,P<0.01) . The results meant TSHR asPNA with NLS structure not only inhibited TSHR mRNA levels in vitro, but also downregulated thyroid TSHR mRNA through i.p. injection. 5. There was no obvious influence of asPNAs on serum antibodies against TSHR. However, T4 value decreased or had a tendency of decrease in mice treated with asPNAs, with reduced thyroid TSHR mRNA levels. In comparison with NLS-scramblePNA treated or normal saline controls, there was no obvious thyroid follicular epithelial cell proliferation and condensed colloid substance in the asPNAs-treated animals. Fewer newborn follicles were seen.CONCLUSION : Ideal Graves' disease mice models are replicated by recombinant expression plasmids pcDNA3.1TOPO/TSHRa, pcDNA3.1TOPO / TSHRc or KLH-TSHR38- 45 and gTSHR immunization. TSHR NLS-asPNA cantreat Graves' disease models to some extent through downregulating TSHRexpression.