Fig 1.
Construction of recombinant plasmid and the expression of human TSHR A subunit via transfected NIH 3T3L1 cells.
Identification of TSHR gene expression by western blot. Cultured 3T3L1 cells were transfected with the recombinant plasmids pcDNA3.1- T289 or the control vector pcDNA3.1 using Lipofectamine 3000 (Invitrogen). 24h after the transfection, western blot assay was performed to detect the expression of TSHR A subunit protein. The first lane of pcDNA3.1-T289 showing more enhanced expression than the second one was due to the different transfection efficiency in different cell plates.
Fig 2.
TSHR genetically immunized mice showed typical features of goiter.
(A) The immunized mice develop the phenotype of goiter. (B) In H&E staining, the TSHR immunized mice showed typical phenotype of hyperthyroidism (×40). Thyroid epithelial cells are tall and columnar and extend as papillary folds into the follicles (arrowed), indications of hypertrophy and hyperplasia. (C) There is increased number of infiltrated mononuclear cells in the thyroids (arrowed, ×200). (D) The immune mice showed significantly increased level of T4 levels in compared with the age-matched controls. The dotted line indicates mean ±3SD for control mice, *p<0.05. (E) The immune mice showed significantly increased level of TRAb compared with the age-matched controls. The TRAb levels were determined by ELISA and expressed as the fold change of OD450 value compared to control sera, *p<0.05.
Fig 3.
TSHR immunized mice showed enlarged extraocular muscle volume.
(A) In vivo MRI scaning of the mouse head showed no sign of proptosis, Contiguous T2-weighted MR images of the head at the horizontal level were acquired using a fast-spin-echo (FSE) sequence (0.6mm thick). Judging from the relative position of the eyeball and the outline of the mouse head, no sign of proptosis were observed in the immune mice compared with control. (B) The enlargement of extraocular muscle volume was observed in in vivo MRI scaning, Contiguous T2-weighted MR images of extraocular muscles of the right eye were acquired at the sagittal level. The orbital muscles (labeled C) are of lower signal intensity than the adjacent cerebrospinal fluid (in white, labeled A). In turn, the orbital muscles are surrounded by the higher signal intensity of the harderian gland (labeled B). (C) Segmentation analysis by Image J software demonstrated there is a significant increase in the extraocular muscle volume, *p<0.05.
Fig 4.
Histological analysis of the orbital tissues from the TSHR immunized mice.
(A) H&E staining of the orbital tissue (×40). Mild lymphocytic infiltration was seen between the extraocular muscle around the optic nerve. (B) Adipose tissue around the optic nerve was increased in immunized animals (×40). (C)Alcian blue-stained section of orbital muscle (×400) showed increased production of HA in retrobulbar tissue in immunized mice.