背景 青光眼滤过手术后人Tenon囊成纤维细胞(HTFs)的增生是滤过手术失败的丰要原因,寻找抑制术后HTFs增生的药物可提高滤过手术的成功率. 目的 观察芹黄素对HTFs体外生长的抑制效应并探讨其作用机制.方法 斜视手术中获取的人眼Tenon囊组织剪成1 mm×1 mm×1 mm的组织块,进行原代培养,免疫荧光法进行细胞波形蛋白检测以鉴定培养的HTFs.取第3～5代HTFs接种于96孔板中,加入0、20、40、80、160 μmol/L芹黄素后继续培养,于24、48、72 h后分别取一块96孔板,在酶联免疫检测仪上检测560 nm波长处各组细胞的吸光度(A560)值,磺基罗丹明B(SRB)法测定细胞的增生能力.分别在培养基中加入40、80、160 μmol/L芹黄素,同时在不同浓度芹黄素组培养基中均加入10 μg/L BrdU,继续培养48 h,在各组同一个视野下分别拍摄荧光显微镜和光学显微镜照片,计算BrdU的标记率,以未添加任何芹黄素(0μmol/L)组为对照组.用Hoechst 33258染色后在荧光显微镜下观察培养细胞的形态学改变,采用流式细胞技术检测细胞的凋亡情况和细胞周期的变化. 结果 培养的细胞生长状态良好,免疫荧光法检测可见细胞对波形蛋白呈阳性反应,为细胞质中均匀的绿色荧光,确定培养的细胞为HTFs.SRB法检测结果显示,HTFs的增生值(A560)随着芹黄素浓度的增加而逐渐下降,同浓度芹黄素组HTFs A560值随着作用时间的延长逐渐下降,差异均有统计学意义(F组别=480.306,P=0.000;F时间 =555.144,P=0.000).0、40、80 μmol/L芹黄素作用HTFs 48 h后BrdU的标记率分别为(87.860±0.632)％、(61.520±4.306)％、(23.480±4.472)％,各组之间的总体比较差异有统计学意义(F=299.347,P=0.000),其中40 μmol/L、80 μ mol/L芹黄素组HTFs BrdU的标记率均明显低于0 μmol/L芹黄素组,差异均有统计学意义(P＜0.05).Hoechse 33258染色发现,不同浓度芹黄素组的细胞数目减少,随着芹黄素浓度的增加,细胞核固缩和变形的细胞数目增加.流式细胞术检测发现,随着芹黄素浓度的增加,G0/G1期细胞的百分数逐渐增加,而S期和G2/M期细胞的百分数逐渐下降,差异均有统计学意义(FG0/G1 =58.621,P=0.000;Fs=32.357,P=0.001；Fc2/M =83.998,P=0.000).0、40、80、160 μmol/L芹黄素作用72 h后,细胞的凋亡率分别为(4.77±0.21)％、(13.24-±1.35)％、(18.33±1.86)％、(31.58±2.77)％,4个组的总体差异有统计学意义(F=204.791,P＜0.05). 结论 芹黄素通过诱导细胞凋亡,并将细胞阻滞于G0/G1期而抑制HTFs的增生,其作用呈剂量和时间依赖性.
揹景 青光眼濾過手術後人Tenon囊成纖維細胞(HTFs)的增生是濾過手術失敗的豐要原因,尋找抑製術後HTFs增生的藥物可提高濾過手術的成功率. 目的 觀察芹黃素對HTFs體外生長的抑製效應併探討其作用機製.方法 斜視手術中穫取的人眼Tenon囊組織剪成1 mm×1 mm×1 mm的組織塊,進行原代培養,免疫熒光法進行細胞波形蛋白檢測以鑒定培養的HTFs.取第3～5代HTFs接種于96孔闆中,加入0、20、40、80、160 μmol/L芹黃素後繼續培養,于24、48、72 h後分彆取一塊96孔闆,在酶聯免疫檢測儀上檢測560 nm波長處各組細胞的吸光度(A560)值,磺基囉丹明B(SRB)法測定細胞的增生能力.分彆在培養基中加入40、80、160 μmol/L芹黃素,同時在不同濃度芹黃素組培養基中均加入10 μg/L BrdU,繼續培養48 h,在各組同一箇視野下分彆拍攝熒光顯微鏡和光學顯微鏡照片,計算BrdU的標記率,以未添加任何芹黃素(0μmol/L)組為對照組.用Hoechst 33258染色後在熒光顯微鏡下觀察培養細胞的形態學改變,採用流式細胞技術檢測細胞的凋亡情況和細胞週期的變化. 結果 培養的細胞生長狀態良好,免疫熒光法檢測可見細胞對波形蛋白呈暘性反應,為細胞質中均勻的綠色熒光,確定培養的細胞為HTFs.SRB法檢測結果顯示,HTFs的增生值(A560)隨著芹黃素濃度的增加而逐漸下降,同濃度芹黃素組HTFs A560值隨著作用時間的延長逐漸下降,差異均有統計學意義(F組彆=480.306,P=0.000;F時間 =555.144,P=0.000).0、40、80 μmol/L芹黃素作用HTFs 48 h後BrdU的標記率分彆為(87.860±0.632)％、(61.520±4.306)％、(23.480±4.472)％,各組之間的總體比較差異有統計學意義(F=299.347,P=0.000),其中40 μmol/L、80 μ mol/L芹黃素組HTFs BrdU的標記率均明顯低于0 μmol/L芹黃素組,差異均有統計學意義(P＜0.05).Hoechse 33258染色髮現,不同濃度芹黃素組的細胞數目減少,隨著芹黃素濃度的增加,細胞覈固縮和變形的細胞數目增加.流式細胞術檢測髮現,隨著芹黃素濃度的增加,G0/G1期細胞的百分數逐漸增加,而S期和G2/M期細胞的百分數逐漸下降,差異均有統計學意義(FG0/G1 =58.621,P=0.000;Fs=32.357,P=0.001；Fc2/M =83.998,P=0.000).0、40、80、160 μmol/L芹黃素作用72 h後,細胞的凋亡率分彆為(4.77±0.21)％、(13.24-±1.35)％、(18.33±1.86)％、(31.58±2.77)％,4箇組的總體差異有統計學意義(F=204.791,P＜0.05). 結論 芹黃素通過誘導細胞凋亡,併將細胞阻滯于G0/G1期而抑製HTFs的增生,其作用呈劑量和時間依賴性.
배경 청광안려과수술후인Tenon낭성섬유세포(HTFs)적증생시려과수술실패적봉요원인,심조억제술후HTFs증생적약물가제고려과수술적성공솔. 목적 관찰근황소대HTFs체외생장적억제효응병탐토기작용궤제.방법 사시수술중획취적인안Tenon낭조직전성1 mm×1 mm×1 mm적조직괴,진행원대배양,면역형광법진행세포파형단백검측이감정배양적HTFs.취제3～5대HTFs접충우96공판중,가입0、20、40、80、160 μmol/L근황소후계속배양,우24、48、72 h후분별취일괴96공판,재매련면역검측의상검측560 nm파장처각조세포적흡광도(A560)치,광기라단명B(SRB)법측정세포적증생능력.분별재배양기중가입40、80、160 μmol/L근황소,동시재불동농도근황소조배양기중균가입10 μg/L BrdU,계속배양48 h,재각조동일개시야하분별박섭형광현미경화광학현미경조편,계산BrdU적표기솔,이미첨가임하근황소(0μmol/L)조위대조조.용Hoechst 33258염색후재형광현미경하관찰배양세포적형태학개변,채용류식세포기술검측세포적조망정황화세포주기적변화. 결과 배양적세포생장상태량호,면역형광법검측가견세포대파형단백정양성반응,위세포질중균균적록색형광,학정배양적세포위HTFs.SRB법검측결과현시,HTFs적증생치(A560)수착근황소농도적증가이축점하강,동농도근황소조HTFs A560치수착작용시간적연장축점하강,차이균유통계학의의(F조별=480.306,P=0.000;F시간 =555.144,P=0.000).0、40、80 μmol/L근황소작용HTFs 48 h후BrdU적표기솔분별위(87.860±0.632)％、(61.520±4.306)％、(23.480±4.472)％,각조지간적총체비교차이유통계학의의(F=299.347,P=0.000),기중40 μmol/L、80 μ mol/L근황소조HTFs BrdU적표기솔균명현저우0 μmol/L근황소조,차이균유통계학의의(P＜0.05).Hoechse 33258염색발현,불동농도근황소조적세포수목감소,수착근황소농도적증가,세포핵고축화변형적세포수목증가.류식세포술검측발현,수착근황소농도적증가,G0/G1기세포적백분수축점증가,이S기화G2/M기세포적백분수축점하강,차이균유통계학의의(FG0/G1 =58.621,P=0.000;Fs=32.357,P=0.001；Fc2/M =83.998,P=0.000).0、40、80、160 μmol/L근황소작용72 h후,세포적조망솔분별위(4.77±0.21)％、(13.24-±1.35)％、(18.33±1.86)％、(31.58±2.77)％,4개조적총체차이유통계학의의(F=204.791,P＜0.05). 결론 근황소통과유도세포조망,병장세포조체우G0/G1기이억제HTFs적증생,기작용정제량화시간의뢰성.
Background Proliferation of the human Tenon capsule fibroblasts(HTFs) is a main cause of failure of filtering surgery.To search the drug of inhibiting the growth of the HTFs is essential for the improvement of successful rate of filtering surgery.Objective The aim of this study was to investigate the inhibitory effect of apigenin on HTFs and its mechanism.Methods Human Tenon capsular tissue was obtained during the strabismus correction surgery.HTFs was primarily cultured using explant method and identified using vimentin by immunochemistry.The 3-5 generation of cells were incubated to 96-well plate.Apigenin of 0,20,40,80,160 μmol/L was added into the medium,respectively,for 24,48,72 hours,and the proliferation of HTFs was detected by sulfonyl chloride (SRB) at the wavelength of 560 nm (A560).Bromodeoxyuridine (BrdU) of 10 μg/L was added to culture the cells for 48 hours to calculate the labeling rate of BrdU.The morphology of the cells was observed using Hoechst 33258 staining,and apoptosis and cells cycle were evaluated by flow cytometry.Results Cultured cells grew well with the positive response for vimentin,showing the green fluorescence in cytoplasm.SRB assay showed that the A560 value was gradually declined with the increase of the dosage of apigenin and prolong of time (Fgroup =480.306,P =0.000 ; Ftime =555.144,P =0.000).The labeling rate after 0,40,80 μmol/L apigenin acted for 48 hours was (87.860 ±0.632)％,(61.520±4.306)％ and (23.480±4.472)％,showing a significant difference among the three groups (F =299.347,P =0.000).The labeling rate of HTFs for BrdU was significantly decreased in the 40 and 80 μmol/L apigenin groups compared with the 0 μmol/L apigenin group (P＜0.05).Hoechse 33258 staining found that the number of the HTFs was gradually decreased and the cell number of karyopyknosis and nuclear deformation was increased with the increase of apigenin dosage.Percentage of cells in G0/G1 phase were raised and that in S and G2/M phase were declined in the higher dosage apigenin group,with a significant difference among the different groups (FG0/G1 =58.621,P=0.000;Fs =32.357,P=0.001 ;FG2/M =83.998,P=0.000).In the 72nd hour after acted by 0,40,80,160 μmol/L apigenin,the apoptosis rate of HTFs was (4.77±0.21) ％,(13.24±1.35)％,(18.33±1.86) ％,(31.58 ± 2.77) ％,respectively,with a statistically significant difference among the four groups (F =204.791,P＜0.05).Conclusions Apigenin restrains the growth of HTFs by evoking G0/G1 cell cycle arrest and inducing apoptosis in a dosage-and time-dependent manner.
