Preliminary Research Of The Role And Mechanism Of LPS In Orbital Fibroblast Proliferation

Purpose: This study used lipopolysaccharide(LPS)to stimulate in vitro cultured CD90+ and CD90-orbital fibroblasts sampled from thyroid associated ophthalmopathy patients,and compared the effect of LPS on the proliferation of orbital fibroblasts with different phenotypes,and the expression of inflammatory cytokines.Also,this study preliminarily discussed the mechanisms by which LPS induce fat proliferation during TAO.Methods: Orbital fibroblasts were sampled from the fat/connective tissue of 6 patients with TAO after primary culture cells were randomly assigned into 7 groups and respectively stimulated by 0 ng/ml,10 ng/ml,50ng/ml,100 ng/ml,200ng/ml,1000 ng/ml and 2000 ng/ml of LPS.The CKK-8(cell counting kit-8)was used to examine the proliferation of orbital fibroblasts in different groups,and the optimal stimulation concentration was thereby determined.Then,flow cytometry was performed to identify CD90-fibroblasts,and CD90-overexpressing lentivirus was constructed and transfected into CD90-cells.Afterwards,cells were divided into group A(empty lentivirus,n=2)and group B(CD90-overexpressinglentivirus,n=2),and each group was stimulated with 1000 ng/ml of LPS.The CCK-8 was used again to measure orbital fibroblast proliferation.RT-PCR and Western-blot were performed to determine the expression of CD90 gene and protein,and RT-PCR was used to measure the expression of IL-6,IL-1β and TNF-α in different groups.Results:(1)Orbital fibroblasts were successfully cultured in vitro,and CD90-cells were successfully isolated.When the concentration of LPS reached 500 ng/ml,the proliferation of fibroblasts was increased by 20%.So 500 ng/mL was determined as the optimal stimulation concentration.(2)CD90-overexpressing lentivirus was successfully constructed and transfected into CD90-orbital fibroblasts.(3)After being stimulated by LPS at different concentration,no significant increase in fibroblast proliferation was detected either by inter-or intra-group comparison.The concentration of LPS showed no dose-effect relationship with the proliferation of orbital fibroblasts,and the difference was insignificant(P>0.05).However,after being stimulated by LPS with the same concentration,CD90-cells showed increased secretion of IL-6,IL-1β and TNF-α,while CD90-overexpressing cells showed decreased secretion of the above cytokines.The differences were significant(P< 0.05).Conclusion: Fat/connective tissue-derived orbital fibroblasts developed inflammatory reactions after LPS stimulation,and cells showed increased expression of IL-6,IL-1β and TNF-α.Orbital fibroblasts expressing CD90-or CD90+ did not show significant difference in proliferation when stimulated by LPS.