| Objective In order to provide new therapy for the thyroid associated ophthalmopathy,the effect of colchicine inducing retroorbital fibroblast (RF) was detect in vitro.We study the morphological changes of RF by microscope,investigate the effect on the proliferation of RF cultured in vitro and determine the expression of TGF-βafter the effect of colchicine.Methods The conjunctival tissue from patients with TAO was cultured and passaged,and used between the third and sixth passage.Then these RF was devided into four COLC groups treated with colchicine of different experinental concentrations(from 10-4mol/L to 10-8mol/L),and the control group were treated by RPMI-1640.After different time points of incubation(24h,48h,72h) with reagent indicated above,The morphological changes of RF were examined under phase-contrast microscope every day and photographed;the cell proliferation was detected by CCK-8 method.Then the former three groups(from 10-4mol/L to 10-6mol/L) were cutured for 48 hours,the apoptosis rate of RF was determined by flow cytomety(FCM).At last,the RF cells were divided into 2 groups,COLC group were expose to COLC at final concentration of 10-4mol/L,control group were treated as above,cultured for 48h,the expression of TGF-βwere observed by PV-6001/6002 PowerVicion Two-step Immunohistochemistry.Result Cultured RF showed the spindle-like in shape and the number of it was significantly increased with the incubation time.The changes of cell morphology occurred as a result of the effect of COLC: except the 10-8mol/L group,other groups showed aging and stopped growing at different level.The CCK-8 methed found that 10-4mol/L,10-5mol/L,10-6mol/L and 10-7mol/L colchicine could increase apoptosis of OFs compared with the control group(P＜0.01) at 24,48,and 72 hours of incubation.The result of FCM analysis had shown that COLC could induce cellular apoptosis and apoptosis induced was dose dependen,the apoptotic rate of COLC groups had significantly increased(p＜0.01), respectively),the percentage of RF in G0/G1 phase was increased and that of G2+M was decreased.By Immunohistochemistry methed we draw the conclution that the colchicin could also decrease the expression of TGF-β,compared to a statistically significant difference(P＜0.05).Conclusion Colchicine can not only induce apoptosis of RF in a time and concentration dependent manner,but also induced apoptosis of RF,and the decline of TGF-βmay be the cause of it.These result imply that Colchicial have potential role in treating the thyroid associated ophthalmopathy.
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