中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
9期
839-844
,共6页
程雪娟%张少斌%林林%王继兵%邹会会
程雪娟%張少斌%林林%王繼兵%鄒會會
정설연%장소빈%림림%왕계병%추회회
翼状胬肉%成纤维细胞%增生%麦考酚酸%p65%核因子-κB
翼狀胬肉%成纖維細胞%增生%麥攷酚痠%p65%覈因子-κB
익상노육%성섬유세포%증생%맥고분산%p65%핵인자-κB
Pterygium%Fibroblast%Proliferation%Macophenolic acid%p65%Nuclear factor-κB
背景 研究表明麦考酚酸(MPA)可下调或抑制与组织增生和炎症相关细胞因子的表达和分泌,进而抑制组织的增生和炎症反应.翼状胬肉是球结膜组织炎性和增生性病变,MPA对翼状胬肉组织的增生是否有抑制作用尚未见报道.目的 研究MPA对翼状胬肉组织成纤维细胞(PFBs)活性的影响,并对其相关机制进行探讨.方法 翼状胬肉切除术中获取的组织经组织块培养法体外培养PFBs,并采用波形蛋白抗体免疫组织化学法进行鉴定.将0、0.125、0.250、0.500、1.000 μmol/LMPA液加入培养孔中,未加入MPA液者作为对照.采用MTT比色法和5-溴脱氧尿嘧啶(BrdU)掺入法观察各组PFBs的生长情况；用Western blot法检测各组PFBs中核因子-κB(NF-κB)、p65及NF-KBα抑制剂(IκB-α)蛋白的表达.结果 培养的细胞为长梭形,呈漩涡状、放射状排列,波形蛋白表达阳性.MTT比色法检测表明,随着MPA浓度的增加,PFBs的增生值(A560)均逐渐下降,5个浓度组间PFBs增生值的总体比较差异有统计学意义(F=42.874,P＜0.01).随着MPA作用时间的延长,PFBs的增生值(A560)逐渐下降,差异有统计学意义(F=26.038,P＜0.01).BrdU 免疫荧光染色发现,随着MPA浓度的增加,PFBs DNA合成量(A560)逐渐下降,5个组间总体比较差异有统计学意义(F=175.279,P＜0.05),各浓度MPA组PFBs DNA合成量与对照组比较均明显下降,差异均有统计学意义(P＜0.05).DAPI免疫荧光染色结果表明,各浓度的MPA作用后均未见PFBs的形态学异常.Western blot法检测显示,作用72 h后,MPA组p65表达量为0.886±0.072,明显低于对照组的1.542±0.124,MPA组PFBs细胞质IκB-α的表达量为2.141±0.305,明显高于对照组的1.559±0.267,差异均有统计学意义(P＜0.05).结论 MPA可抑制PFBs的生长,其作用机制可能与其抑制PFBs的NF-κB通路有关.
揹景 研究錶明麥攷酚痠(MPA)可下調或抑製與組織增生和炎癥相關細胞因子的錶達和分泌,進而抑製組織的增生和炎癥反應.翼狀胬肉是毬結膜組織炎性和增生性病變,MPA對翼狀胬肉組織的增生是否有抑製作用尚未見報道.目的 研究MPA對翼狀胬肉組織成纖維細胞(PFBs)活性的影響,併對其相關機製進行探討.方法 翼狀胬肉切除術中穫取的組織經組織塊培養法體外培養PFBs,併採用波形蛋白抗體免疫組織化學法進行鑒定.將0、0.125、0.250、0.500、1.000 μmol/LMPA液加入培養孔中,未加入MPA液者作為對照.採用MTT比色法和5-溴脫氧尿嘧啶(BrdU)摻入法觀察各組PFBs的生長情況；用Western blot法檢測各組PFBs中覈因子-κB(NF-κB)、p65及NF-KBα抑製劑(IκB-α)蛋白的錶達.結果 培養的細胞為長梭形,呈漩渦狀、放射狀排列,波形蛋白錶達暘性.MTT比色法檢測錶明,隨著MPA濃度的增加,PFBs的增生值(A560)均逐漸下降,5箇濃度組間PFBs增生值的總體比較差異有統計學意義(F=42.874,P＜0.01).隨著MPA作用時間的延長,PFBs的增生值(A560)逐漸下降,差異有統計學意義(F=26.038,P＜0.01).BrdU 免疫熒光染色髮現,隨著MPA濃度的增加,PFBs DNA閤成量(A560)逐漸下降,5箇組間總體比較差異有統計學意義(F=175.279,P＜0.05),各濃度MPA組PFBs DNA閤成量與對照組比較均明顯下降,差異均有統計學意義(P＜0.05).DAPI免疫熒光染色結果錶明,各濃度的MPA作用後均未見PFBs的形態學異常.Western blot法檢測顯示,作用72 h後,MPA組p65錶達量為0.886±0.072,明顯低于對照組的1.542±0.124,MPA組PFBs細胞質IκB-α的錶達量為2.141±0.305,明顯高于對照組的1.559±0.267,差異均有統計學意義(P＜0.05).結論 MPA可抑製PFBs的生長,其作用機製可能與其抑製PFBs的NF-κB通路有關.
배경 연구표명맥고분산(MPA)가하조혹억제여조직증생화염증상관세포인자적표체화분비,진이억제조직적증생화염증반응.익상노육시구결막조직염성화증생성병변,MPA대익상노육조직적증생시부유억제작용상미견보도.목적 연구MPA대익상노육조직성섬유세포(PFBs)활성적영향,병대기상관궤제진행탐토.방법 익상노육절제술중획취적조직경조직괴배양법체외배양PFBs,병채용파형단백항체면역조직화학법진행감정.장0、0.125、0.250、0.500、1.000 μmol/LMPA액가입배양공중,미가입MPA액자작위대조.채용MTT비색법화5-추탈양뇨밀정(BrdU)참입법관찰각조PFBs적생장정황；용Western blot법검측각조PFBs중핵인자-κB(NF-κB)、p65급NF-KBα억제제(IκB-α)단백적표체.결과 배양적세포위장사형,정선와상、방사상배렬,파형단백표체양성.MTT비색법검측표명,수착MPA농도적증가,PFBs적증생치(A560)균축점하강,5개농도조간PFBs증생치적총체비교차이유통계학의의(F=42.874,P＜0.01).수착MPA작용시간적연장,PFBs적증생치(A560)축점하강,차이유통계학의의(F=26.038,P＜0.01).BrdU 면역형광염색발현,수착MPA농도적증가,PFBs DNA합성량(A560)축점하강,5개조간총체비교차이유통계학의의(F=175.279,P＜0.05),각농도MPA조PFBs DNA합성량여대조조비교균명현하강,차이균유통계학의의(P＜0.05).DAPI면역형광염색결과표명,각농도적MPA작용후균미견PFBs적형태학이상.Western blot법검측현시,작용72 h후,MPA조p65표체량위0.886±0.072,명현저우대조조적1.542±0.124,MPA조PFBs세포질IκB-α적표체량위2.141±0.305,명현고우대조조적1.559±0.267,차이균유통계학의의(P＜0.05).결론 MPA가억제PFBs적생장,기작용궤제가능여기억제PFBs적NF-κB통로유관.
Background Studies showed that macophenolic acid (MPA)down-regulates and inhibits the expression and secretion of tissue growth factor and inflammatory factor,and further impacts the proliferation and inflammation process.Pterygium is an inflammatory and proliferative lesion.Whether MPA has an inhibitory effect on pterygium is unclear.Objective This study was to investigate the antifibrotic effects of macophenolic acid on pterygium fibroblasts(PFBs) in vitro and discuss its mechanism.Methods Pterygium tissue was obtained from pterygium patient during the surgery.PFBs were cultured using explants and identified with vimentin immunohistochemisty.0,0.125,0.250,0.500,1.000 μmol/L MPA were added into the culture medium,respectively,and the cells were cultured in the medium without MPA as the control group.MTT colorimetry was used to find the optimization effective concentration of MPA and evaluate their inhibitory effect on PFBs,and BrdU fluorescence staining was used to assess the growth statue of PFBs.Expressions of nuclear factor-κB(NF-κB),p65 and inhibitor of NF-κB-α(IκB-α) in the cells were detected by Western blot.Results The cells was spindle in shape 3 days after cultured and showed the vortex and radial arrangement with the positive response to vimentin.With the increase of MPA,the proliferative value of PFBs (A560)showed gradually decline,with a significant difference among the five groups (F =42.874,P＜0.01).In addition,the proliferative value of PFBs (A560) significantly lowed as the prolong of MPA active time(F=26.038,P＜0.01).BrdU fluorescence staining showed a significant decrease of DNA synthesis of PFBs with the elevation of MPA dose among the five groups(F=175.279,P＜0.05),and the A560of PFBs DNA synthesis in different concentrations of MPA groups was lower than that of the control group (all at P＜0.05).No apoptotic and necrotic cell was found after MPA action by DAPI staining.The expression level of p65 in the PFBs was 0.886±0.072 and 1.542±0.124 in the MPA group and the control group,indicating a declined value in the MPA group(P＜0.05).However,the expression value of IκB-α in the cytoplasm PFBs was significantly higher in the MPA group compared with the control group(2.141 ±0.305 vs.1.559±0.267) (P＜0.05).Conclusions MPA has an inhibitory effect on the growth of PFBs,which probably is related to the arresting of NF-κB pathway.
