中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
10期
907-912
,共6页
甲状腺相关眼病%前脂肪细胞%成纤维细胞%脂多糖%过氧化物酶体增生物激活受体-γ%环氧合酶-2
甲狀腺相關眼病%前脂肪細胞%成纖維細胞%脂多糖%過氧化物酶體增生物激活受體-γ%環氧閤酶-2
갑상선상관안병%전지방세포%성섬유세포%지다당%과양화물매체증생물격활수체-γ%배양합매-2
Thyroid-associated ophthalmopathy%Preadipocytes%Fibroblast%Lipopolysaccharide%Peroxisome proliferator activated receptor-γ%Cyclooxygenase-2
背景 脂肪增生和炎症反应是甲状腺相关眼病(TAO)活动期的两大主要病理过程,美伐他汀被证明有抑制前脂肪细胞分化的作用.目的 观察美伐他汀对TAO来源眼眶前脂肪细胞分化的作用,及对环氧合酶-2(COX-2)与过氧化物酶体增生物激活受体-γ(PPAR-γ)表达的影响,探讨其对脂多糖(LPS)诱导炎症反应及TAO眼眶前脂肪细胞分化的调控作用.方法 从4例TAO患者眼眶减压术中获取眼眶球后脂肪组织,用组织块培养法体外培养TAO眼眶成纤维细胞,为观察美伐他汀对TAO炎症反应过程中COX-2的作用,将培养细胞分为5个组,A组用10000μg/L的LPS,B、C、D组采用1000 μg/L LPS分别联合5、10、20 μmol/L美伐他汀,E组不加任何干预药物作为对照,均作用8h.观察美伐他汀对眶脂肪细胞分化后的作用,将A组再亚分为A1～A6组,1000 μg/L LPS作用8h后,加入诱导液诱导各组细胞向脂肪细胞分化,A1组不加任何干预药物,A2、A3、A4组分别在分化全程中加入5、10、20 μmol/L美伐他汀,A5、A6组分别在分化的第4天与第8天加入10μmol/L美伐他汀直至分化结束.采用油红0染色法测定分化后脂肪细胞的相对含量,应用Western blot法和逆转录聚合酶链反应(RT-PCR)法检测成纤维细胞中COX-2和PPAR-γ蛋白及其mRNA的表达,用酶联免疫吸附试验(ELISA)检测细胞培养上清液中前列腺素E2(PGE2)的表达.结果 B、C、D组眶成纤维细胞中COX-2蛋白及其mRNA的表达和PGE2的分泌水平均较A组明显降低(P＜0.05).随着美伐他汀浓度的升高,COX-2蛋白及其mRNA的表达和PGE2的分泌水平均逐渐降低,各组间总体差异均有统计学意义(F=228.380、101.745、1586.881,P＜O.05).E组COX-2蛋白及其mRNA的表达和PGE2的分泌水平较A、B、C组明显减弱,差异均有统计学意义(P＜0.05),但与D组比较差异无统计学意义(P＞0.05).A1、A2、A3、A4组前脂肪细胞分化后吸光度(A492)值逐渐下降,分化后的细胞PPAR-γ蛋白及其mRNA表达均依次下降,组间两两比较差异均有统计学意义(P＜0.05)；A1与A6组间的A492值、PPAR-γ蛋白及其mRNA的表达差异均无统计学意义(P＞0.05)；A1、A5、A3组的A492值及PPAR-γ蛋白及其mRNA表达均依次下降,组间比较差异均有统计学意义(P＜0.05).结论 美伐他汀以剂量依赖的方式抑制LPS激活的TAO眼眶成纤维细胞中COX-2表达、TAO眼眶前脂肪细胞的分化、PPAR-γ的表达和PGE2的分泌,前脂肪细胞分化的早期抑制作用更强.
揹景 脂肪增生和炎癥反應是甲狀腺相關眼病(TAO)活動期的兩大主要病理過程,美伐他汀被證明有抑製前脂肪細胞分化的作用.目的 觀察美伐他汀對TAO來源眼眶前脂肪細胞分化的作用,及對環氧閤酶-2(COX-2)與過氧化物酶體增生物激活受體-γ(PPAR-γ)錶達的影響,探討其對脂多糖(LPS)誘導炎癥反應及TAO眼眶前脂肪細胞分化的調控作用.方法 從4例TAO患者眼眶減壓術中穫取眼眶毬後脂肪組織,用組織塊培養法體外培養TAO眼眶成纖維細胞,為觀察美伐他汀對TAO炎癥反應過程中COX-2的作用,將培養細胞分為5箇組,A組用10000μg/L的LPS,B、C、D組採用1000 μg/L LPS分彆聯閤5、10、20 μmol/L美伐他汀,E組不加任何榦預藥物作為對照,均作用8h.觀察美伐他汀對眶脂肪細胞分化後的作用,將A組再亞分為A1～A6組,1000 μg/L LPS作用8h後,加入誘導液誘導各組細胞嚮脂肪細胞分化,A1組不加任何榦預藥物,A2、A3、A4組分彆在分化全程中加入5、10、20 μmol/L美伐他汀,A5、A6組分彆在分化的第4天與第8天加入10μmol/L美伐他汀直至分化結束.採用油紅0染色法測定分化後脂肪細胞的相對含量,應用Western blot法和逆轉錄聚閤酶鏈反應(RT-PCR)法檢測成纖維細胞中COX-2和PPAR-γ蛋白及其mRNA的錶達,用酶聯免疫吸附試驗(ELISA)檢測細胞培養上清液中前列腺素E2(PGE2)的錶達.結果 B、C、D組眶成纖維細胞中COX-2蛋白及其mRNA的錶達和PGE2的分泌水平均較A組明顯降低(P＜0.05).隨著美伐他汀濃度的升高,COX-2蛋白及其mRNA的錶達和PGE2的分泌水平均逐漸降低,各組間總體差異均有統計學意義(F=228.380、101.745、1586.881,P＜O.05).E組COX-2蛋白及其mRNA的錶達和PGE2的分泌水平較A、B、C組明顯減弱,差異均有統計學意義(P＜0.05),但與D組比較差異無統計學意義(P＞0.05).A1、A2、A3、A4組前脂肪細胞分化後吸光度(A492)值逐漸下降,分化後的細胞PPAR-γ蛋白及其mRNA錶達均依次下降,組間兩兩比較差異均有統計學意義(P＜0.05)；A1與A6組間的A492值、PPAR-γ蛋白及其mRNA的錶達差異均無統計學意義(P＞0.05)；A1、A5、A3組的A492值及PPAR-γ蛋白及其mRNA錶達均依次下降,組間比較差異均有統計學意義(P＜0.05).結論 美伐他汀以劑量依賴的方式抑製LPS激活的TAO眼眶成纖維細胞中COX-2錶達、TAO眼眶前脂肪細胞的分化、PPAR-γ的錶達和PGE2的分泌,前脂肪細胞分化的早期抑製作用更彊.
배경 지방증생화염증반응시갑상선상관안병(TAO)활동기적량대주요병리과정,미벌타정피증명유억제전지방세포분화적작용.목적 관찰미벌타정대TAO래원안광전지방세포분화적작용,급대배양합매-2(COX-2)여과양화물매체증생물격활수체-γ(PPAR-γ)표체적영향,탐토기대지다당(LPS)유도염증반응급TAO안광전지방세포분화적조공작용.방법 종4례TAO환자안광감압술중획취안광구후지방조직,용조직괴배양법체외배양TAO안광성섬유세포,위관찰미벌타정대TAO염증반응과정중COX-2적작용,장배양세포분위5개조,A조용10000μg/L적LPS,B、C、D조채용1000 μg/L LPS분별연합5、10、20 μmol/L미벌타정,E조불가임하간예약물작위대조,균작용8h.관찰미벌타정대광지방세포분화후적작용,장A조재아분위A1～A6조,1000 μg/L LPS작용8h후,가입유도액유도각조세포향지방세포분화,A1조불가임하간예약물,A2、A3、A4조분별재분화전정중가입5、10、20 μmol/L미벌타정,A5、A6조분별재분화적제4천여제8천가입10μmol/L미벌타정직지분화결속.채용유홍0염색법측정분화후지방세포적상대함량,응용Western blot법화역전록취합매련반응(RT-PCR)법검측성섬유세포중COX-2화PPAR-γ단백급기mRNA적표체,용매련면역흡부시험(ELISA)검측세포배양상청액중전렬선소E2(PGE2)적표체.결과 B、C、D조광성섬유세포중COX-2단백급기mRNA적표체화PGE2적분비수평균교A조명현강저(P＜0.05).수착미벌타정농도적승고,COX-2단백급기mRNA적표체화PGE2적분비수평균축점강저,각조간총체차이균유통계학의의(F=228.380、101.745、1586.881,P＜O.05).E조COX-2단백급기mRNA적표체화PGE2적분비수평교A、B、C조명현감약,차이균유통계학의의(P＜0.05),단여D조비교차이무통계학의의(P＞0.05).A1、A2、A3、A4조전지방세포분화후흡광도(A492)치축점하강,분화후적세포PPAR-γ단백급기mRNA표체균의차하강,조간량량비교차이균유통계학의의(P＜0.05)；A1여A6조간적A492치、PPAR-γ단백급기mRNA적표체차이균무통계학의의(P＞0.05)；A1、A5、A3조적A492치급PPAR-γ단백급기mRNA표체균의차하강,조간비교차이균유통계학의의(P＜0.05).결론 미벌타정이제량의뢰적방식억제LPS격활적TAO안광성섬유세포중COX-2표체、TAO안광전지방세포적분화、PPAR-γ적표체화PGE2적분비,전지방세포분화적조기억제작용경강.
Background Inflammation and adipogenesis are two parallel processes with increasing activity in severe thyroid-associated ophthalmopathy(TAO),and mevastatin was proved to have the inhibiting effect on the differentiation of adipose.Objective The aim of this work was to investigate the effects of mevastatin on the expression of cyclooxygenase-2(COX-2)and peroxisome proliferator activated receptor-γ(PPAR-γ)and differentiation of TAO-derived orbital preadipocytes,and explore its modulation effects on lipopolysaccharide(LPS)induced inflammation and the differentiation of TAO-derived orbital preadipocytes in vitro.Methods The retroorbital adipose tissue was obtained from 4 TAO patients during the surgery.The orbital fibroblasts were cultured from orbital adipose tissues using explant culture method.To study the suppressing effect of mevastatin on inflammatory response,cultured cells were divided into 5 groups.The 1000 μg/L LPS orbital fibroblasts were stimulated for 8 hours in group A,and 1000 μg/L LPS combined with 5 μmol/L,10 μmoL/L or 20 μmoL/L mevastatin were used respectively for the substitute in the group B,group C and group D.The orbital fibroblasts in group E were cultured routinely without any intervention as control.To observe the inhibiting effect of mevastatin on the differentiation of adipose,the group A were then subdivided into group A1-A6.After 1000 μg/L LPS was used to treat the cells for 8 hours,the ceils were induced to differentiate into adipocytes.All orbital preadipocytes from A1 to A6 were stimulated to differentiate into mature adipocytes with cocktail differentiation medium for a 10-day duration.During the procedure,group A2,A3 and A4 were interfered with 5,10 or 20 μmol/L mevastatin,and in the group A5 and A6,10 μmol/L mevastatin were added at the fourth day or eighth day.Intracellular fat accumulation in differentiated adipocytes was determined by oil red O staining.The absorption(A492 nm)was measured in the ceils by enzyme-linked immunosorbent assay(ELISA).Expression of COX-2 and PPAR-γ mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR),and the expression of COX-2 and PPAR-γ protein was detected by Westernblot.The level of PGE2 in the supernatant was detected by ELISA.Results The expression of COX-2 protein and mRNA as well as the PGE2 levels in B,C,D group decreased markedly in comparison with those in A group(P＜0.05).With the increase of mevastatin concentration,the expression of COX-2 protein and mRNA as well as the PGE2 levels in B,C,D groups decreased successively(F =228.380,101.745,1586.881,P＜0.05).The expression of COX-2 protein and mRNA and PGE2 levels in E group were lower significantly than those in A,B and C groups(P＜0.05),but no significant differences were found between E group and D group(P＞0.05).The A492 value and the expressions of PPAR-γ protein and mRNA in differentiated cells showed the successively decrease in A1-A4 group with the elevation of mevastatin concentration(P＜0.05),and the evidently decreased A492 value and the expressions of PPAR-γ protein and mRNA also were seen in A1 and A5 groups compared with A3 group(P ＜ 0.05).Conclusions Mevastatin inhibits LPS-induced COX-2 expression,PPAR-γ expression,PGE2 secretion and differentiation of TAO-derived orbital fibroblasts in vitro in dose-dependent manner.Mevastatin plays these effect more prominently in early stage of adipocytes differentiation.
