Antisense PNA Inhibiting The Expression Of TSHR And The Probable Therapeutic Effect On Graves' Disease

Objective:1. To study the effect of antisense PNA on thyrotropin receptor (TSHR) on FRTL cells membrane2. To investigate the probable therapeutic effect of PNA on Graves' disease Methods:1. The part of cells1.1 We designed two kinds of asPNA which were hybridized to the TSHR mRNA named as NLS-asPNA1, NLS-asPNA2, to investigate the cellular uptake by microscopy, both PNAs were linked by their N-terminus to fluorescein(FL). We also designed a control PNA named as NLS-scrPNA and NLS-scrPNA—FL.1.2 To observe the cellular uptake of PNA, the cells were seeded and divided into 4 groups, named as A, B, C, D, then the cells of B, C, D groups were exposed for a certain time to NLS-asPNA1-FL , NLS-asPNA2-FL and NLS-scrPNA-FL, after 48h, the cells were anlysed using a Leica microscopy.1.3 The cells were seeded in 96-well plate and treated with PNA to investigate if the PNAs have the toxicity. The PNAs were added to the medium 24h after seeding, OD was measured at 24, 48, 72h using MTT, the data are expressed in terms of T/C( OD of PNA-treated cells/OD of control cells)1.4 We detected the effect of NLS-asPNA on TSHR mRNA by Realtime RT-PCR. The cells were divided into 3 groups( A, B, C), which were addedNLS-asPNAl, NLS-asPNA2, NLS-scrPNA in the medium respectively, and the final concentration of PNA was lOuM. Then total cellular RNA was extracted after Oh, 12h, 24h, 48h and reverse-transcripted to cDNA. We measured the levels of TSHR mRNA by Realtime RT-PCR kit. 2. The part of animals2.1 Guinea pig TSHR (called gTSHR) was extracted from adipose cells, and measured its concentration by CBG.2.2 Synthesized TSHR aa36-42, and conjugate with KLH ,called KLH-TSHR36-42.2.3 The mice were divided into 4 groups, named as A ^ B > C ^ D. gTSHR+KLH-TSHR36-42 were injected into abdomen of mice in above different groups(A^ B> C) each 2 weeks and the mice saline injected as control (D), after 6 weeks, the mice of group A> B^ C were injected NLS-asPNAl, NLS-scrPNA and saline respectively, The levels of total T4 (TT4), thyroid stimulating hormone receptor antibody (TRAb), thyrotropin receptor stimulating antibody (TSAb) in serum of mice were measured. The animals were killed on 10 weeks, and two sides of thyroid were taken and the level of TSHR mRNA in thyroid tissue and the pathological changes in mice thyroid were detected.Results:1. The part of cells1.1 We found that all NLS-PNA were taken up by the cells using microscopy.1.2 We measured the T/C and found that, there was no significant change of different times.1.3 The results of Realtime RT-PCR showed that, lOuM NLS-asPNAl could reduce the expression of TSHR mRNA, the levels of mRNA were 43% and 16.98% ofthe control at 24 and 48h. lOuM NLS-asPNAl could also reduce the expression of TSHR mRNA, and the levels of mRNA were 61.66% and 33.11% of the control at 24 and 48h, both had a time-dependent character. But there is no significant change of NLS-scrPNA. 2. The part of animals2.1 The concentration of gTSHR was 59mg/dL using CBG.2.2 The levels of TRAb: Comparing with the data in the same group on day 0, the levels of TRAb were increased in 6 and 10 weeks in the groups of A> B, C, but there is no difference in the group of D.2.3 The levels of TSAb were decreased in the 6 and 10 weeks in the group of A, comparing with 0 weeks, and have significant difference, but it was increased in the group B and C and no significant difference, the levels of TSAb of group D didn't have significant changes.2.4 In the group A ■> B and C, the levels of T4 was increased in the 6w, comparing with Ow and have significant difference. After 4w, the levels of T4 had a significant decrease in group A but not happened in group B and C.2.5 The results of Realtime RT-PCR showed that, the levels of TSHR mRNA of group A was lower than C, and had significant difference between A and C, but there was no significant difference between B and C.2.6 The thyroid follicle of the group of B and C was shown some pathological changes such as augmentation, epithelial proliferation and condensed colloid material , and had so many neogenesis follicle, but the thyroid follicle of the group A had no significant augmentation and condensed colloid material.Conclusions:NLS-asPNA could be taken up by the cells , reduced the expression of TSHR, and had a time-depended character, it also could relieve the pathogenetic condition of Graves' disease, so it has instructive effect on GD.