The Role Of MiR-146a In The Regulation Of IL-6 And IL-8 In The Orbital Fibroblaste Of Thyroid Associated Ophthalmopathy

OBJECTIVE: To up-regulate and down-regulate the expression level of miR-146 a of the orbital fibroblast in vitro,then,to discuss role of miR-146 a in the regulation of IL-6 and IL-8 in the orbital fibroblasts of thyroid associated ophthalmopathy.METHODS: This study set up the experimental groups and control groups.The otrbital connective tissue samples of experimental groups,from April 2016 to May 2017,came from six patients with thyroid related ophthalmopathy who were presented the first affiliated hospital of guangxi medical university and underwent orbital decompression or strabismus diorthosis due to exposure keratitis,oppressive optic neuropathy or restrictive extraocular myopathy.The orbital connective tissue samples of control groups were taken from six patients with eyeball atrophy or other reason enucleation and prosthesis implantation caused by ocular trauma.The orbital connective tissue samples were used to set up primary culture and were identified by immunohistochemistry.Using lentivirus with target gene to transfect into orbital fibroblasts,the expression level of miR-146 a of orbital fibroblasts were up-regulate or down-regulate,then the expression levels of IL-6 and IL-8 were detected by ELISA.The statistical analysis was done with the Statistical Program for the Social Sciences(SPSS,version 20.0,SPSS Inc.,Chicago).P Values <0.05 were considered as statistically significant.RESULTS:(1)orbital fibroblasts from patients with thyroid associated ophthalmopathy and normal human were relatively easy to grow in vitro,and there were no significant difference in morphology.The growth curve showed that cultured cells had normal proliferation characteristics as an s-shape.Immunohistochemical staining results show that: vimentin(+),desrmin(-),myoglobin(-),keralin(-),S100(-).(2)When MOI=50,lentivirus transfection conditions were optimal,transfection efficiency >90%,and proper concentration of puromycin screening is 3.5?g/ml.(3)there was a significant increase in il-6 and il-8 levels orbital fibroblasts from thyroid associated ophthalmopathy and the results were statistically different from those in the normal group(P<0.05).In the NC-mimic146 a group,the levels of il-6 and il-8 in the supernatant were significantly decreased,and the results were statistically different compare to the control group(P<0.05).In the NC-inhibitor146 a group,the level of IL-8 in the supernatant increased significantly,the results was statistically different compare to the control group(P<0.05),however,IL-6 level have no statistical difference(P = 0.306).In the TAO-mimic146 a group,the levels of il-6 and il-8 in the supernatant were significantly decreased,and the results were statistically different compare to the control group(P<0.05).At the same time,in the TAO-inhibitor146 a group,the levels of il-6 and il-8 in the supernatant were significantly increased,and the results were statistically different compare to the control group(P < 0.05).CONCLUSIONS: Primary cell culture techniques can be used to obtain purified orbital fibroblasts.Under appropriate conditions,the lentiviruses with target gene can transfect orbital fibroblasts in vitro,over expression of mi R-146 a inhibits orbital fibroblasts in the expression of IL-6 and IL-8,and inhibition of miR-146 a can promote orbital fibroblasts in the expression of IL-6 and IL-8.It is suggested that miR-146 a may be related to the development of thyroid associated ophthalmopathy.