中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2011年
2期
156-161
,共6页
脂多糖类%Graves眼病%环氧化酶2%PPARγ%细胞分化
脂多糖類%Graves眼病%環氧化酶2%PPARγ%細胞分化
지다당류%Graves안병%배양화매2%PPARγ%세포분화
Lipopolysaccharides%Graves ophthalmopathy%Cyclooxygenase2%PPAR gamma%Cell differentiation
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。
目的 觀察脂多糖誘導炎癥反應對甲狀腺相關眼病( TAO)眼眶前脂肪細胞分化的影響，探討TAO眼眶脂肪增殖的髮病機製。方法 實驗研究。眼眶組織取TAO行開眶減壓術的患者。將體外培養的TAO眼眶成纖維細胞，分為A組與B組，A組採用1 mg/L的脂多糖作用8h，然後常規用誘導分化液Ⅰ和Ⅱ誘導直至分化結束，B組僅用誘導分化液Ⅰ和Ⅱ誘導至分化結束。採用油紅O染色法測定分化後脂肪細胞相對含量，逆轉錄聚閤酶鏈反應法與Western blot法檢測環氧化酶2( COX2)與過氧化物酶體增殖物激活受體γ(PPARγ) mRNA與蛋白的錶達，酶聯免疫吸附實驗檢測上清液中前列腺素E2( PG E2)的錶達。兩樣本均數比較採用t檢驗，多樣本比較採用單因素方差分析，兩兩比較採用SNK檢驗。結果 分化後油紅O染色顯示兩組細胞形態上無明顯差異。A組細胞吸光度(A)值(1.02±0.08)較B組(0.74±0.06)明顯升高，差異具有統計學意義(t=8.502，P=0.000)。A組細胞分化後PPARγ mRNA( 1.74±0.19)與蛋白(0.47 ±0.04)的錶達分彆較分化前PPARγ mRNA(0.30±0.07)與蛋白(0.08±0.02)的錶達明顯增彊(P＜0.05)；B組細胞分化後PPARγ mRNA(1.15 ±0.18)與蛋白(0.35±0.03)的錶達分彆較分化前PPARγ mRNA(0.13 ±0.04)與蛋白(0.03 ±0.01)的錶達明顯增彊(P＜0.05)；A組細胞分化後COX2 mRNA (0.28±0.07)與蛋白(0.24±0.03)的錶達分彆較分化前COX2 mRNA( 1.54 ±0.10)與蛋白(0.49±0.03)的錶達明顯減弱(P＜0.05)；B組細胞分化後COX2 mRNA的錶達（0.08±0.03）較分化前(0.19±0.07)明顯減弱(P＜0.05)，而COX2蛋白的錶達(0.01±0.01)與分化前(0.03±0.01)比較差異無統計學意義(P＞0.05)。分化後A組細胞上清液中PGE2分泌水平(208.43±15.06)ng/L較分化前(898.75±21.09)ng/L顯著降低(P＜0.05)；分化後B組細胞上清液中PGE2分泌水平(30.61±5.75) ng/L與分化前（35.75±4.09）ng/L比較，差異無統計學意義(P＞0.05)。誘導分化前A組細胞COX2 mRNA與蛋白的錶達及PGE2的分泌較B組明顯增彊(P＜0.05)，而PPARγ mRNA與蛋白的錶達亦彊于B組(P＜0.05)，分化後A組細胞COX2及PPARγmRNA與蛋白的錶達、PGE2的分泌均彊于B組(P＜0.05)。結論 脂多糖可誘導TAO眼眶前脂肪細胞產生炎癥反應，使其炎癥標誌物COX2及其控製閤成的PGE2錶達增彊，併使脂肪生成關鍵轉錄因子PPARγ錶達上調，分化後PPARγ錶達顯著增彊，而COX2與PGE2的錶達明顯降低。
목적 관찰지다당유도염증반응대갑상선상관안병( TAO)안광전지방세포분화적영향，탐토TAO안광지방증식적발병궤제。방법 실험연구。안광조직취TAO행개광감압술적환자。장체외배양적TAO안광성섬유세포，분위A조여B조，A조채용1 mg/L적지다당작용8h，연후상규용유도분화액Ⅰ화Ⅱ유도직지분화결속，B조부용유도분화액Ⅰ화Ⅱ유도지분화결속。채용유홍O염색법측정분화후지방세포상대함량，역전록취합매련반응법여Western blot법검측배양화매2( COX2)여과양화물매체증식물격활수체γ(PPARγ) mRNA여단백적표체，매련면역흡부실험검측상청액중전렬선소E2( PG E2)적표체。량양본균수비교채용t검험，다양본비교채용단인소방차분석，량량비교채용SNK검험。결과 분화후유홍O염색현시량조세포형태상무명현차이。A조세포흡광도(A)치(1.02±0.08)교B조(0.74±0.06)명현승고，차이구유통계학의의(t=8.502，P=0.000)。A조세포분화후PPARγ mRNA( 1.74±0.19)여단백(0.47 ±0.04)적표체분별교분화전PPARγ mRNA(0.30±0.07)여단백(0.08±0.02)적표체명현증강(P＜0.05)；B조세포분화후PPARγ mRNA(1.15 ±0.18)여단백(0.35±0.03)적표체분별교분화전PPARγ mRNA(0.13 ±0.04)여단백(0.03 ±0.01)적표체명현증강(P＜0.05)；A조세포분화후COX2 mRNA (0.28±0.07)여단백(0.24±0.03)적표체분별교분화전COX2 mRNA( 1.54 ±0.10)여단백(0.49±0.03)적표체명현감약(P＜0.05)；B조세포분화후COX2 mRNA적표체（0.08±0.03）교분화전(0.19±0.07)명현감약(P＜0.05)，이COX2단백적표체(0.01±0.01)여분화전(0.03±0.01)비교차이무통계학의의(P＞0.05)。분화후A조세포상청액중PGE2분비수평(208.43±15.06)ng/L교분화전(898.75±21.09)ng/L현저강저(P＜0.05)；분화후B조세포상청액중PGE2분비수평(30.61±5.75) ng/L여분화전（35.75±4.09）ng/L비교，차이무통계학의의(P＞0.05)。유도분화전A조세포COX2 mRNA여단백적표체급PGE2적분비교B조명현증강(P＜0.05)，이PPARγ mRNA여단백적표체역강우B조(P＜0.05)，분화후A조세포COX2급PPARγmRNA여단백적표체、PGE2적분비균강우B조(P＜0.05)。결론 지다당가유도TAO안광전지방세포산생염증반응，사기염증표지물COX2급기공제합성적PGE2표체증강，병사지방생성관건전록인자PPARγ표체상조，분화후PPARγ표체현저증강，이COX2여PGE2적표체명현강저。
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.
