| Purpose:To compare the cell morphological and ultrastructue, surface antigen expression and adipogenic differentiation rate of orbital preadipocytes between patients with thyroid-associated ophthalmopathy (TAO) and health adults,the cells were cultured in vitro.To analysis the expression of human leucocyte antigen-DR(HLA-DR) in these two kinds of cells,investigate the effects of Interferon-γ(IFN-γ) and Interleukin-6(IL-6) on the expression of HLA-DR in the differentiated adipocytes.To study the effects of pioglitazone and GW9662 on orbital preadipocytes proliferation,differentiation and expression of HLA-DR.Methods:Orbital preadipocytes from TAO and normal subjects were cultured and committed to differentiate.Cell morphological and ultrastructue were compared through optical and electron microscopy. Flow cytometry was used to analysis the expression of cell surface antigens.HLA-DR expression was examined with immunohistochemical technique.The rate of adipogenic differentiation was compared of the orbital preadipocytes between TAO and health adults.The effects of HLA-DR expression on IL-6 and IFN-γduring differentiation were detected through Flow cytometry and real time RT-PCR.The effects of pioglitazone and GW9662 on preadipocytes proliferation were examined by the methods of MTT.The effects on preadipocytes differentiation were examined by oil red O staining.The effects of HLA-DR expression on pioglitazone and GW9662 during differentiation were detected through flow cytometry and real time RT-PCR.Results:Tissue culture methods and enzyme - digesting methods are both reliable for the primary cell culture and differentiate effectively of human preadipocytes.Abundant of fatty drop bodies and fatty deposition were found under electron microscopy in TAO orbital preadipocytes.Positive expression of HLA-DR was examined in TAO orbital preadipocytes,the geometric mean fluorescent intensity(14.93±0.73) was significantly different compared to health adults(3.55±0.60) (P＜0.05).Positive expression of HLA-DR was examined in TAO orbital preadipocytes cytoplasm and cytomembrane,while negative expression in health adults with immunohistochemical technique.Adipogenic differentiation rate was significantly up rated in TAO orbital preadipocytes(21.3%) compared with health adults(12.1%)(P＜0.05) The geometric mean fluorescent intensity(26.15±0.64) was significantly up rated compared to before differentiated(14.93±0.73 ) in TAO orbital preadipocytes by flow cytometry(P＜0.05).The expression of HLA-DR was 9.3folds up rated after differentiation measured by real time RT-PCR in TAO orbital preadipocytes.IFN-γstimulated the expression of HLA-DR in TAO and health adult orbital preadipocytes.The geometric mean fluorescent intensities(45.26±1.28 and 14.59±0.62) were significantly up rated compared to control groups(26.15±0.64 and 3.28±0.58) by flow cytometry(P＜0.05).The expression of HLA-DR was significantly up rated compared to control group after differentiation under IFN-γmeasured by real time RT-PCR in TAO orbital preadipocytes (P＜0.05).IL-6 could not stimulate the expression of HLA-DR in TAO and health adult orbital preadipocytes.The geometric mean fluorescent intensities(27.28±1.30 and 4.35±0.26) were not significantly different compared to control groups(26.15±0.64 and 3.28±0.58) by flow cytometry(P＞0.05).The expression of HLA-DR was not significantly different compared to control group after differentiation under IL-6 measured by real time RT-PCR in TAO orbital preadipocytes(P＞0.05). The OD values of 0.1μmol/L,1μmol/L,10μmol/L and 50μmol/L pioglitazone were(0.046±0.005),(0.051±0.004),(0.060±0.003)and (0.020±0.004),the later three groups were significantly different compared to control group(0.046±0.004) after 48h in TAO orbital preadipocytes(P＜0.05).The OD values of 0.1μmol/L,1μmol/L,10 μmol/L and 50μmol/L pioglitazone were(0.047±0.006),(0.050±0.003),(0.056±0.004) and(0.018±0.005),the later three groups were significantly different compared to control group(0.046±0.004) after 48h in health adults orbital preadipocytes(P＜0.05).When the concentration attained 1μmol/L,Pioglitazone stimulated human orbit preadipocytes proliferation concentration depended,It had no significantly difference between the orbit preadipocytes in TAO and health adults(P＞0.05).When the concentration attained 50μmol/L,the orbit preadipocytes of TAO and health adults were partly detached from the cultural bottles,the proliferation of the cells were obviously inhibited. The OD values of 0.1μmol/L,1μmol/L,10μmol/L and 50μmol/L pioglitazone were(1.286±0.082),(1.489±0.109),(1.758±0.115) and (0.206±0.028) after differentiation,the later three groups were significantly different compared to control group(1.238±0.090) in TAO orbital preadipocytes(P＜0.05).The OD values of 0.1μmol/L,1μmol/L,10μmol/L and 50μmol/L pioglitazone were(1.053±0.083),(1.212±0.091),(1.329±0.108) and(0.186±0.019) after differentiation, the later three groups were significantly different compared to control group(1.046±0.096) in health adults orbital preadipocytes(P＜0.05). When the concentration attained 1μmol/L,Pioglitazone stimulated human orbit preadipocytes differentiation concentration depended,It could stimulate the orbit preadipocytes differentiation of patients with TAO more obviously than health adults(P＜0.05).When the concentration attained 50μmol/L,the orbit preadipocytes of TAO and health adults were partly detached from the cell culture bottles,the differentiation of the cells were obviously inhibited.The OD values of 0.1μmol/L,1μmol/L,10μmol/L and 50μmol/L GW9662 were(1.235±0.106),(1.031±0.062),(0.608±0.050) and(0.156±0.013) after differentiation,the later three groups were significantly different compared to control group(1.238±0.090) in TAO orbital preadipocytes (P＜0.05).The OD values of 0.1μmol/L,1μmol/L,10μmol/L and 50μmol/L pioglitazone were(1.045±0.085),(0.942±0.073),(0.605±0.041) and(0.131±0.011) after differentiation,the later three groups were significantly different compared to control group(1.046±0.096) in health adults orbital preadipocytes(P＜0.05).When the concentration attained 1μmol/L,GW9662 inhibited human orbit preadipocytes differentiation concentration depended,It could inhibit the orbit preadipocytes differentiation of patients with TAO more obviously than health adults(P＜0.05).When the concentration attained 50μmol/L,the orbit preadipocytes of TAO and health adults were partly detached from the cell culture bottles,the differentiation of the cells were obviously inhibited.The OD values of 10μmol/L pioglitazone and 10μmol/L GW9662 were 0.635±0.083 and 0.630±0.084,which were significantly different compared to control group(1.238±0.090 and 1.046±0.096) and pioglitazone group(1.758±0.115 and 1.329±0.108)after differentiation in TAO and health adults orbital preadipocytes(P＜0.05). 10μmol/L pioglitazone stimulated the expression of HLA-DR in TAO orbital preadipocytes after differentiation.The geometric mean fluorescent intensity(34.19±1.09) was significantly up rated compared to control groups(26.15±0.64 ) by flow cytometry(P＜0.05).The expression of HLA-DR was 3.1folds up rated after differentiation measured by real time RT-PCR in TAO orbital preadipocytes.10μmol/L GW9662 deceased the expression of HLA-DR in TAO orbital preadipocytes after differentiation.The geometric mean fluorescent intensity(17.34±0.94) was significantly down rated compared to control groups(26.15±0.64) by flow cytometry(P＜0.05).The expression of HLA-DR was 0.3fold down rated after differentiation measured by real time RT-PCR in TAO orbital preadipocytes.10μmol/L pioglitazone and 10μmol/L GW9662 could not stimulate the expression of HLA-DR in health adults orbital preadipocytes after differentiation.The geometric mean fluorescent intensity(4.03±0.57 and 3.26±0.31) was not significantly differentiated compared to control groups(3.28±0.58) by flow cytometry(P＞0.05).Conclusions:There are differences of morphology and biology in the orbital preadipocytes between TAO and health adults.Abundant of fatty drop bodies,high rate of adipogenic differentiation and expression of HLA-DR are found in TAO orbital preadipocytes.It might participate in the expansion of the orbital contents and modulation of orbital immune reaction.IFN-γcan stimulate the expression of HLA-DR in orbital preadipocytes after differentiation,which can increase the local immune reaction and inflammation.Orbital preadipocytes may then act as antigen presenting cells to participate the regulation of the immune procession. IL-6 can't stimulate the expression of HLA-DR in orbital preadipocytes after differentiation.It may not be involved in the regulation of TAO orbital inflammatory lesions.Pioglitazone can stimulate the proliferation and differentiation on orbital preadipocytes,while also can enhance the expression of HLA-DR after differentiation in TAO orbital preadipocytes. It may induce and worsen the disease.GW9662 can inhibit the proliferation and differentiation on orbital preadipocytes,induce the expression of HLA-DR after differentiation in TAO orbital preadipocytes. It may be a new way to prevent and treat TAO.
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