甲状腺相关眼病眼眶前脂肪细胞分化与HLA-DR相关因素的研究
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摘要
目的:利用体外培养的甲状腺相关眼病(thyroid associatedophthalmopathy,TAO)和正常人眼眶前脂肪细胞,比较其超微结构、表面抗原、脂肪细胞转化率等指标的差异;了解眼眶前脂肪细胞人类白细胞抗原DR(human leucocyte antigen-DR,HLA-DR)的表达,研究干扰素γ(Interferon-γ,IFN-γ)、白细胞介素6(Interleukin-6,IL-6)对前脂肪细胞分化过程中HLA-DR表达的影响;探讨匹格列酮和GW9662对眼眶前脂肪细胞增殖和分化的作用和分化过程中HLA-DR表达的影响。
方法:原代培养TAO和正常人眼眶前脂肪细胞并体外诱导分化。光镜和电镜下比较两种细胞的形态学差异,流式细胞术检测细胞表面抗原的表达,免疫组化检测细胞HLA-DR的表达,脂肪细胞转化率比较两者的脂肪分化能力。流式细胞术和实时荧光定量RT-PCR研究IL-6、IFN-γ对细胞分化过程中HLA-DR表达的调节作用。MTT法测定匹格列酮、GW9662对前脂肪细胞增殖的影响,油红0染色定量法测定匹格列酮、GW9662对前脂肪细胞分化的影响,流式细胞术和实时荧光定量RT-PCR研究匹格列酮、GW9662对细胞分化过程中HLA-DR表达的调节作用。
结果:组织块法和酶消化法均能从组织中获取相同生物活性的眼眶前脂肪细胞,并成功向脂肪细胞诱导分化。电镜下观察TAO眼眶前脂肪细胞含有丰富的脂滴小体和脂质沉积。流式细胞术检测TAO眼眶前脂肪细胞表达HLA-DR阳性,几何平均荧光强度为14.93±0.73,与正常人(3.55±0.60)比较,差异有显著性意义(P＜0.05)。免疫组化法检测TAO眼眶前脂肪细胞胞浆和胞膜表达HLA-DR阳性,而正常人表达阴性。体外诱导分化后,TAO眼眶前脂肪细胞的脂肪细胞转化率(21.3%)较正常人(12.1%)增高,两者间差异有显著性意义(P＜0.05)。流式细胞术检测TAO眼眶前脂肪细胞分化后HLA-DR表达增强,几何平均荧光强度为26.15±0.64,与分化前(14.93±0.73)相比,差异有显著性意义(P＜0.05);实时荧光定量RT-PCR检测TAO眼眶前脂肪细胞分化后HLA-DR的表达较分化前增强了9.3倍。加入IFN-γ刺激,TAO和正常人眼眶前脂肪细胞分化后HLA-DR的表达均有增强,流式细胞术检测其几何平均荧光强度分别为45.26±1.28和14.59±0.62,与对照组(26.15±0.64和3.28±0.58)比较,差异有显著性意义(P＜0.05);实时荧光定量RT-PCR检测IFN-γ刺激下TAO眼眶前脂肪细胞分化后HLA-DR表达较分化前增强了25.5倍,与对照组(9.3倍)比较,差异有显著性意义(P＜0.05)。加入IL-6刺激,TAO和正常人眼眶前脂肪细胞分化后HLA-DR的表达均无明显变化,流式细胞术检测其几何平均荧光强度分别为和27.28±1.30和4.35±0.26,与对照组(26.15±0.64和3.28±0.58)比较,差异无显著性意义(P＞0.05);实时荧光定量RT-PCR检测IL-6刺激下TAO眼眶前脂肪细胞分化后HLA-DR表达较分化前增强了9.6倍,与对照组(9.3倍)比较,差异无显著性意义(P＞0.05)。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的匹格列酮作用48h后,TAO眼眶前脂肪细胞的OD值分别为0.046±0.005、0.051±0.004、0.060±0.003和0.020±0.004,后三组与对照组(0.046±0.004)相比,差异有显著性意义(P＜0.05)。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的匹格列酮作用48h后,正常人眼眶前脂肪细胞的OD值分别为0.047±0.006、0.050±0.003、0.056±0.004和0.018±0.005,后三组与对照组(0.047±0.005)相比,差异有显著性意义(P＜0.05)。当浓度达到1μmol/L,匹格列酮以浓度依赖性促进TAO和正常人眼眶前脂肪细胞的增殖,对两者增殖的作用差异无显著性意义(P＞0.05);当浓度达到50μmol/L,TAO和正常人眼眶前脂肪细胞呈现部分脱壁,细胞增殖受到明显抑制。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的匹格列酮干预诱导分化后,TAO眼眶前脂肪细胞的OD值分别为1.286±0.082、1.489±0.109、1.758±0.115和0.206±0.028,后三组与对照组(1.238±0.090)相比,差异有显著性意义(P＜0.05)。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的匹格列酮干预诱导分化后,正常人眼眶前脂肪细胞的OD值分别为1.053±0.083、1.212±0.091、1.329±0.108和0.186±0.019,后三组与对照组(1.046±0.096)相比,差异有显著性意义(P＜0.05)。当浓度达到1μmol/L,匹格列酮以浓度依赖性促进TAO和正常人眼眶前脂肪细胞的分化,且对TAO眼眶前脂肪细胞有更强的促分化作用,对两者的促分化作用差异有显著性意义(P＜0.05);当浓度达到50μmol/L,TAO和正常人眼眶前脂肪细胞呈现部分脱壁,细胞分化受到明显抑制。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的GW9662干预诱导分化后,TAO眼眶前脂肪细胞的OD值分别为1.235±0.106、1.031±0.062、0.608±0.050和0.156±0.013,后三组与对照组(1.238±0.090)相比,差异有显著性意义(P＜0.05)。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的GW9662干预诱导分化后,正常人眼眶前脂肪细胞的OD值分别为1.045±0.085、0.942±0.073、0.605±0.041和0.131±0.011,后三组与对照组(1.046±0.096)相比,差异有显著性意义(P＜0.05)。当浓度达到1μmol/L,GW9662以浓度依赖性抑制TAO和正常人眼眶前脂肪细胞的分化,且对TAO眼眶前脂肪细胞的分化有更强的抑制作用,对两者的抑制分化作用差异有显著性意义(P＜0.05);当浓度达到50μmol/L,TAO和正常人眼眶前脂肪细胞呈现部分脱壁,细胞分化受到明显抑制。用10μmol/L/L浓度的匹格列酮和10μmol/L浓度的GW9662混合液干预前脂肪细胞分化后,TAO和正常人眼眶前脂肪细胞的OD值分别为0.635±0.083和0.630±0.084,与匹格列酮组(1.758±0.115和1.329±0.108)及对照组(1.238±0.090和1.046±0.096)相比,差异有显著性意义(P＜0.05)。10μmol/L浓度的匹格列酮刺激下,TAO眼眶前脂肪细胞分化后HLA-DR的表达有增强,流式细胞术检测其几何平均荧光强度为34.19±1.09,与对照组(26.15±0.64)比较,差异有显著性意义(P＜0.05);实时荧光定量RT-PCR检测10μmol/L浓度匹格列酮刺激下,TAO眼眶前脂肪细胞分化后HLA-DR表达较对照组增强了3.1倍。10μmol/L浓度的GW9662刺激下,TAO眼眶前脂肪细胞分化后HLA-DR的表达有减弱,流式细胞术检测其几何平均荧光强度为1 7.34±0.94,与对照组(26.15±0.64)比较,差异有显著性意义(P＜0.05);实时荧光定量RT-PCR检测10μmol/L浓度GW9662刺激下,TAO眼眶前脂肪细胞分化后HLA-DR表达为对照组的0.3倍。10μmol/L浓度匹格列酮和10μmol/L浓度GW9662不能刺激正常人眼眶前脂肪细胞分化后HLA-DR的表达,流式细胞术检测其几何平均荧光强度为4.03±0.57和3.26±0.31,与对照组(3.28±0.58)比较,差异无显著性意义(P＞0.05)。
结论:TAO和正常人眼眶前脂肪细胞存在着形态学和生物学上的差异,前者富含脂滴小体,脂肪转化率高,表达HLA-DR,且在分化后表达增强,有可能参与眼眶脂肪组织的扩增和眶内免疫反应的调节。IFN-γ能刺激眼眶前脂肪细胞分化后HLA-DR的表达,因而上调局部免疫反应或炎症反应,使眼眶前脂肪细胞成为一种抗原呈递细胞,参与该病的自身免疫过程。IL-6不能刺激眼眶前脂肪细胞分化后HLA-DR的表达,说明未经该途径参与TAO眶内炎症病变的调节。匹格列酮可以促进眼眶前脂肪细胞的增殖和分化,刺激TAO眼眶前脂肪细胞分化过程中HLA-DR的表达,从而上调局部免疫反应,有可能诱发和加重TAO的发生。GW9662可抑制眼眶前脂肪细胞的分化,拮抗匹格列酮对眼眶前脂肪细胞的促分化作用,并能抑制TAO眼眶前脂肪细胞分化过程中HLA-DR的表达,从而抑制局部免疫反应,有望成为预防和治疗TAO的新方法。
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.
方法:原代培养TAO和正常人眼眶前脂肪细胞并体外诱导分化。光镜和电镜下比较两种细胞的形态学差异,流式细胞术检测细胞表面抗原的表达,免疫组化检测细胞HLA-DR的表达,脂肪细胞转化率比较两者的脂肪分化能力。流式细胞术和实时荧光定量RT-PCR研究IL-6、IFN-γ对细胞分化过程中HLA-DR表达的调节作用。MTT法测定匹格列酮、GW9662对前脂肪细胞增殖的影响,油红0染色定量法测定匹格列酮、GW9662对前脂肪细胞分化的影响,流式细胞术和实时荧光定量RT-PCR研究匹格列酮、GW9662对细胞分化过程中HLA-DR表达的调节作用。
结果:组织块法和酶消化法均能从组织中获取相同生物活性的眼眶前脂肪细胞,并成功向脂肪细胞诱导分化。电镜下观察TAO眼眶前脂肪细胞含有丰富的脂滴小体和脂质沉积。流式细胞术检测TAO眼眶前脂肪细胞表达HLA-DR阳性,几何平均荧光强度为14.93±0.73,与正常人(3.55±0.60)比较,差异有显著性意义(P＜0.05)。免疫组化法检测TAO眼眶前脂肪细胞胞浆和胞膜表达HLA-DR阳性,而正常人表达阴性。体外诱导分化后,TAO眼眶前脂肪细胞的脂肪细胞转化率(21.3%)较正常人(12.1%)增高,两者间差异有显著性意义(P＜0.05)。流式细胞术检测TAO眼眶前脂肪细胞分化后HLA-DR表达增强,几何平均荧光强度为26.15±0.64,与分化前(14.93±0.73)相比,差异有显著性意义(P＜0.05);实时荧光定量RT-PCR检测TAO眼眶前脂肪细胞分化后HLA-DR的表达较分化前增强了9.3倍。加入IFN-γ刺激,TAO和正常人眼眶前脂肪细胞分化后HLA-DR的表达均有增强,流式细胞术检测其几何平均荧光强度分别为45.26±1.28和14.59±0.62,与对照组(26.15±0.64和3.28±0.58)比较,差异有显著性意义(P＜0.05);实时荧光定量RT-PCR检测IFN-γ刺激下TAO眼眶前脂肪细胞分化后HLA-DR表达较分化前增强了25.5倍,与对照组(9.3倍)比较,差异有显著性意义(P＜0.05)。加入IL-6刺激,TAO和正常人眼眶前脂肪细胞分化后HLA-DR的表达均无明显变化,流式细胞术检测其几何平均荧光强度分别为和27.28±1.30和4.35±0.26,与对照组(26.15±0.64和3.28±0.58)比较,差异无显著性意义(P＞0.05);实时荧光定量RT-PCR检测IL-6刺激下TAO眼眶前脂肪细胞分化后HLA-DR表达较分化前增强了9.6倍,与对照组(9.3倍)比较,差异无显著性意义(P＞0.05)。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的匹格列酮作用48h后,TAO眼眶前脂肪细胞的OD值分别为0.046±0.005、0.051±0.004、0.060±0.003和0.020±0.004,后三组与对照组(0.046±0.004)相比,差异有显著性意义(P＜0.05)。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的匹格列酮作用48h后,正常人眼眶前脂肪细胞的OD值分别为0.047±0.006、0.050±0.003、0.056±0.004和0.018±0.005,后三组与对照组(0.047±0.005)相比,差异有显著性意义(P＜0.05)。当浓度达到1μmol/L,匹格列酮以浓度依赖性促进TAO和正常人眼眶前脂肪细胞的增殖,对两者增殖的作用差异无显著性意义(P＞0.05);当浓度达到50μmol/L,TAO和正常人眼眶前脂肪细胞呈现部分脱壁,细胞增殖受到明显抑制。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的匹格列酮干预诱导分化后,TAO眼眶前脂肪细胞的OD值分别为1.286±0.082、1.489±0.109、1.758±0.115和0.206±0.028,后三组与对照组(1.238±0.090)相比,差异有显著性意义(P＜0.05)。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的匹格列酮干预诱导分化后,正常人眼眶前脂肪细胞的OD值分别为1.053±0.083、1.212±0.091、1.329±0.108和0.186±0.019,后三组与对照组(1.046±0.096)相比,差异有显著性意义(P＜0.05)。当浓度达到1μmol/L,匹格列酮以浓度依赖性促进TAO和正常人眼眶前脂肪细胞的分化,且对TAO眼眶前脂肪细胞有更强的促分化作用,对两者的促分化作用差异有显著性意义(P＜0.05);当浓度达到50μmol/L,TAO和正常人眼眶前脂肪细胞呈现部分脱壁,细胞分化受到明显抑制。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的GW9662干预诱导分化后,TAO眼眶前脂肪细胞的OD值分别为1.235±0.106、1.031±0.062、0.608±0.050和0.156±0.013,后三组与对照组(1.238±0.090)相比,差异有显著性意义(P＜0.05)。浓度为0.1μmol/L、1μmol/L、10μmol/L和50μmol/L的GW9662干预诱导分化后,正常人眼眶前脂肪细胞的OD值分别为1.045±0.085、0.942±0.073、0.605±0.041和0.131±0.011,后三组与对照组(1.046±0.096)相比,差异有显著性意义(P＜0.05)。当浓度达到1μmol/L,GW9662以浓度依赖性抑制TAO和正常人眼眶前脂肪细胞的分化,且对TAO眼眶前脂肪细胞的分化有更强的抑制作用,对两者的抑制分化作用差异有显著性意义(P＜0.05);当浓度达到50μmol/L,TAO和正常人眼眶前脂肪细胞呈现部分脱壁,细胞分化受到明显抑制。用10μmol/L/L浓度的匹格列酮和10μmol/L浓度的GW9662混合液干预前脂肪细胞分化后,TAO和正常人眼眶前脂肪细胞的OD值分别为0.635±0.083和0.630±0.084,与匹格列酮组(1.758±0.115和1.329±0.108)及对照组(1.238±0.090和1.046±0.096)相比,差异有显著性意义(P＜0.05)。10μmol/L浓度的匹格列酮刺激下,TAO眼眶前脂肪细胞分化后HLA-DR的表达有增强,流式细胞术检测其几何平均荧光强度为34.19±1.09,与对照组(26.15±0.64)比较,差异有显著性意义(P＜0.05);实时荧光定量RT-PCR检测10μmol/L浓度匹格列酮刺激下,TAO眼眶前脂肪细胞分化后HLA-DR表达较对照组增强了3.1倍。10μmol/L浓度的GW9662刺激下,TAO眼眶前脂肪细胞分化后HLA-DR的表达有减弱,流式细胞术检测其几何平均荧光强度为1 7.34±0.94,与对照组(26.15±0.64)比较,差异有显著性意义(P＜0.05);实时荧光定量RT-PCR检测10μmol/L浓度GW9662刺激下,TAO眼眶前脂肪细胞分化后HLA-DR表达为对照组的0.3倍。10μmol/L浓度匹格列酮和10μmol/L浓度GW9662不能刺激正常人眼眶前脂肪细胞分化后HLA-DR的表达,流式细胞术检测其几何平均荧光强度为4.03±0.57和3.26±0.31,与对照组(3.28±0.58)比较,差异无显著性意义(P＞0.05)。
结论:TAO和正常人眼眶前脂肪细胞存在着形态学和生物学上的差异,前者富含脂滴小体,脂肪转化率高,表达HLA-DR,且在分化后表达增强,有可能参与眼眶脂肪组织的扩增和眶内免疫反应的调节。IFN-γ能刺激眼眶前脂肪细胞分化后HLA-DR的表达,因而上调局部免疫反应或炎症反应,使眼眶前脂肪细胞成为一种抗原呈递细胞,参与该病的自身免疫过程。IL-6不能刺激眼眶前脂肪细胞分化后HLA-DR的表达,说明未经该途径参与TAO眶内炎症病变的调节。匹格列酮可以促进眼眶前脂肪细胞的增殖和分化,刺激TAO眼眶前脂肪细胞分化过程中HLA-DR的表达,从而上调局部免疫反应,有可能诱发和加重TAO的发生。GW9662可抑制眼眶前脂肪细胞的分化,拮抗匹格列酮对眼眶前脂肪细胞的促分化作用,并能抑制TAO眼眶前脂肪细胞分化过程中HLA-DR的表达,从而抑制局部免疫反应,有望成为预防和治疗TAO的新方法。
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.
引文
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