脂多糖诱导炎症反应对甲状腺相关眼病眼眶前脂肪细胞分化的影响

目的 观察脂多糖诱导炎症反应对甲状腺相关眼病( TAO)眼眶前脂肪细胞分化的影响,探讨TAO眼眶脂肪增殖的发病机制。方法 实验研究。眼眶组织取TAO行开眶减压术的患者。将体外培养的TAO眼眶成纤维细胞,分为A组与B组,A组采用1 mg/L的脂多糖作用8h,然后常规用诱导分化液Ⅰ和Ⅱ诱导直至分化结束,B组仅用诱导分化液Ⅰ和Ⅱ诱导至分化结束。采用油红O染色法测定分化后脂肪细胞相对含量,逆转录聚合酶链反应法与Western blot法检测环氧化酶2( COX2)与过氧化物酶体增殖物激活受体γ(PPARγ) mRNA与蛋白的表达,酶联免疫吸附实验检测上清液中前列腺素E2( PG E2)的表达。两样本均数比较采用t检验,多样本比较采用单因素方差分析,两两比较采用SNK检验。结果 分化后油红O染色显示两组细胞形态上无明显差异。A组细胞吸光度(A)值(1.02±0.08)较B组(0.74±0.06)明显升高,差异具有统计学意义(t=8.502,P=0.000)。A组细胞分化后PPARγ mRNA( 1.74±0.19)与蛋白(0.47 ±0.04)的表达分别较分化前PPARγ mRNA(0.30±0.07)与蛋白(0.08±0.02)的表达明显增强(P＜0.05);B组细胞分化后PPARγ mRNA(1.15 ±0.18)与蛋白(0.35±0.03)的表达分别较分化前PPARγ mRNA(0.13 ±0.04)与蛋白(0.03 ±0.01)的表达明显增强(P＜0.05);A组细胞分化后COX2 mRNA (0.28±0.07)与蛋白(0.24±0.03)的表达分别较分化前COX2 mRNA( 1.54 ±0.10)与蛋白(0.49±0.03)的表达明显减弱(P＜0.05);B组细胞分化后COX2 mRNA的表达（0.08±0.03）较分化前(0.19±0.07)明显减弱(P＜0.05),而COX2蛋白的表达(0.01±0.01)与分化前(0.03±0.01)比较差异无统计学意义(P＞0.05)。分化后A组细胞上清液中PGE2分泌水平(208.43±15.06)ng/L较分化前(898.75±21.09)ng/L显著降低(P＜0.05);分化后B组细胞上清液中PGE2分泌水平(30.61±5.75) ng/L与分化前（35.75±4.09）ng/L比较,差异无统计学意义(P＞0.05)。诱导分化前A组细胞COX2 mRNA与蛋白的表达及PGE2的分泌较B组明显增强(P＜0.05),而PPARγ mRNA与蛋白的表达亦强于B组(P＜0.05),分化后A组细胞COX2及PPARγmRNA与蛋白的表达、PGE2的分泌均强于B组(P＜0.05)。结论 脂多糖可诱导TAO眼眶前脂肪细胞产生炎症反应,使其炎症标志物COX2及其控制合成的PGE2表达增强,并使脂肪生成关键转录因子PPARγ表达上调,分化后PPARγ表达显著增强,而COX2与PGE2的表达明显降低。

Effects of LPS-induced inflammation on differentiation of orbital pre-adipocytes in thyroid-associated ophthalmopathy

Objective To investigate the effects of lipopolysaccharide ( LPS)-induced inflammation on the differentiation of orbital pre-adipocytes in thyroid-associated ophthalmopathy (TAO) and to explore the mechanism of orbital adipose proliferation in TAO. Methods Orbital adipose tissues were obtained from patients with TAO undergoing orbital decompression surgery. The orbital fibroblssts cultured from orbital adipose tissues were divided into group A and group B. In group A, orbital pre-adipocytes were incubated inculture medium containing 1 mg/L LPS for 8 hours before stimulated to differentiate into mature adipocytes with Differentiation Medium Ⅰ and Ⅱ. No LPS or other intervention was used in group B before induced to differentiate into mature adipocyte with Differentiation Medium Ⅰ and Ⅱ. Intracellular fat accumulation in differentiated adipocytes was determined by oil red 0 staining and the expression of cyclooxygenase2 (COX2) and peroxisome proliferators activated receptor γ(PPARγ) mRNA of both groups were detected by RT-PCR. Protein expression of COX2 and PPARγ in both groups was detected by Western-blot and the secretion of PGE2 in the supernatant was detected by ELISA. Results COX2 expression and secretion of PGE2 in differentiated cells of both groups were significantly decreased compared with pre-differentiation ( P ＜ 0. 05 ), while PPARγ mRNA and protein expression enhanced significantly ( P ＜ 0. 05 ). COX2 mRNA and protein expression and secretion of PGE2 of pre-differentiation cells in group A was significantly increased compared with group B (P ＜0. 05 ), while PPARγ mRNA and protein expression in group A was also stronger than those in the group B (P ＜ 0. 05 ). COX2 and PPARγ mRNA and protein expression and secretion of PGE2 of differentiated cells in group A were greater than those in group B ( P ＜ 0. 05 ).Conclusion LPS can induce inflammatory response of orbital preadipocytes in TAO and enhance the expression of COX2, PPARγ and PGE2. The expression of PPARγ is enhanced significantly while the expression of COX2 and PGE2 is attenuated markedly when the orbital pre-adipocytes differentiated into adipocytes.