背景 甲状腺相关眼病(TAO)患者的眼眶成纤维细胞(OFs)在眼部的增生性和炎症性反应中发挥重要作用,寻求抑制OFs增生的药物对TAO的防治具有重要意义.已有研究表明,秋水仙碱具有抗组织纤维化的作用,但其对TAO患者OFs的作用及其机制研究较少. 目的 研究秋水仙碱对体外培养的OFs增生的影响. 方法 将3例TAO患者行开眶减压术中获取的球后结缔组织用组织块培养法进行培养和鉴定,取3～8代的传代细胞进行体外实验.分别将1×10-5、1×10-7、1×10-6、1×10-5、1×10-4 mol/L秋水仙碱加入含质量分数10％小牛血清的RPMI 1640培养基中孵育OFs 24、48和72 h,用无秋水仙碱的RPMI 1640培养基孵育细胞作为对照,采用细胞计数试剂盒-8(CCK-8)检测各组细胞450 nm处的吸光度(A450)值,评估OFs的生长情况和各浓度组秋水仙碱对OFs的抑制率.此外,分别在培养基中加入1×10-6、1×10-5、1×10-4 mol/L秋水仙碱作用48 h,采用流式细胞术及应用MCYCLE软件进行分析,用百分率记录亚二倍体细胞数据及细胞周期各时期数据,检测各浓度药物组及无药物培养基组OFs的凋亡率和细胞周期的分布；采用免疫组织化学染色法测定转化生长因子-β(TGF-β)在OFs中的表达,以评价不同浓度秋水仙碱对OFs分泌TGF-β功能的影响.结果 培养和传代的OFs呈梭形,波形蛋白免疫化学染色为阳性反应.1×10-4、1×10-5、1 ×10-6、1×10-7、1×10-8mol/L秋水仙碱作用24、48和72 h,随着秋水仙碱浓度的增加,OFs的增生值(A450)逐渐下降,差异有统计学意义(F浓度=62.004,P＜0.05)；此外,随着秋水仙碱作用时间的增加,A450值逐渐下降,差异有统计学意义(F时间=459.582,P＜0.05)；随着秋水仙碱浓度的增加,其对OFs生长的抑制率逐渐升高.对照组、1×10-6、1×10-5、1×10-4 mol/L秋水仙碱组OFs凋亡率分别为(1.73±0.15)％、(21.04±4.56)％、(31.84±6.21)％和(35.32±5.56)％,4个组间差异有统计学意义(F=83.905,P＜0.05),各组间比较差异均有统计学意义(P＜0.05)；随着秋水仙碱浓度的增加,G2+M期细胞百分数逐渐下降,差异有统计学意义(F=20.443,P＜0.05).免疫组织化学法检测发现,在融合状态前的OFs可以分泌TGF-β,阳性率为(97.60±2.09)％,而1×10-4 mol/L秋水仙碱作用后OFs密度明显减低,部分OFs阳性率为(44.43±3.96)％,差异有统计学意义(t=65.330,P＜0.05). 结论 秋水仙碱可能通过减少OFs分泌TGF-β抑制TAO患者OFs的增生并诱导OFs的凋亡,其作用呈时间和剂量依赖性.
揹景 甲狀腺相關眼病(TAO)患者的眼眶成纖維細胞(OFs)在眼部的增生性和炎癥性反應中髮揮重要作用,尋求抑製OFs增生的藥物對TAO的防治具有重要意義.已有研究錶明,鞦水仙堿具有抗組織纖維化的作用,但其對TAO患者OFs的作用及其機製研究較少. 目的 研究鞦水仙堿對體外培養的OFs增生的影響. 方法 將3例TAO患者行開眶減壓術中穫取的毬後結締組織用組織塊培養法進行培養和鑒定,取3～8代的傳代細胞進行體外實驗.分彆將1×10-5、1×10-7、1×10-6、1×10-5、1×10-4 mol/L鞦水仙堿加入含質量分數10％小牛血清的RPMI 1640培養基中孵育OFs 24、48和72 h,用無鞦水仙堿的RPMI 1640培養基孵育細胞作為對照,採用細胞計數試劑盒-8(CCK-8)檢測各組細胞450 nm處的吸光度(A450)值,評估OFs的生長情況和各濃度組鞦水仙堿對OFs的抑製率.此外,分彆在培養基中加入1×10-6、1×10-5、1×10-4 mol/L鞦水仙堿作用48 h,採用流式細胞術及應用MCYCLE軟件進行分析,用百分率記錄亞二倍體細胞數據及細胞週期各時期數據,檢測各濃度藥物組及無藥物培養基組OFs的凋亡率和細胞週期的分佈；採用免疫組織化學染色法測定轉化生長因子-β(TGF-β)在OFs中的錶達,以評價不同濃度鞦水仙堿對OFs分泌TGF-β功能的影響.結果 培養和傳代的OFs呈梭形,波形蛋白免疫化學染色為暘性反應.1×10-4、1×10-5、1 ×10-6、1×10-7、1×10-8mol/L鞦水仙堿作用24、48和72 h,隨著鞦水仙堿濃度的增加,OFs的增生值(A450)逐漸下降,差異有統計學意義(F濃度=62.004,P＜0.05)；此外,隨著鞦水仙堿作用時間的增加,A450值逐漸下降,差異有統計學意義(F時間=459.582,P＜0.05)；隨著鞦水仙堿濃度的增加,其對OFs生長的抑製率逐漸升高.對照組、1×10-6、1×10-5、1×10-4 mol/L鞦水仙堿組OFs凋亡率分彆為(1.73±0.15)％、(21.04±4.56)％、(31.84±6.21)％和(35.32±5.56)％,4箇組間差異有統計學意義(F=83.905,P＜0.05),各組間比較差異均有統計學意義(P＜0.05)；隨著鞦水仙堿濃度的增加,G2+M期細胞百分數逐漸下降,差異有統計學意義(F=20.443,P＜0.05).免疫組織化學法檢測髮現,在融閤狀態前的OFs可以分泌TGF-β,暘性率為(97.60±2.09)％,而1×10-4 mol/L鞦水仙堿作用後OFs密度明顯減低,部分OFs暘性率為(44.43±3.96)％,差異有統計學意義(t=65.330,P＜0.05). 結論 鞦水仙堿可能通過減少OFs分泌TGF-β抑製TAO患者OFs的增生併誘導OFs的凋亡,其作用呈時間和劑量依賴性.
배경 갑상선상관안병(TAO)환자적안광성섬유세포(OFs)재안부적증생성화염증성반응중발휘중요작용,심구억제OFs증생적약물대TAO적방치구유중요의의.이유연구표명,추수선감구유항조직섬유화적작용,단기대TAO환자OFs적작용급기궤제연구교소. 목적 연구추수선감대체외배양적OFs증생적영향. 방법 장3례TAO환자행개광감압술중획취적구후결체조직용조직괴배양법진행배양화감정,취3～8대적전대세포진행체외실험.분별장1×10-5、1×10-7、1×10-6、1×10-5、1×10-4 mol/L추수선감가입함질량분수10％소우혈청적RPMI 1640배양기중부육OFs 24、48화72 h,용무추수선감적RPMI 1640배양기부육세포작위대조,채용세포계수시제합-8(CCK-8)검측각조세포450 nm처적흡광도(A450)치,평고OFs적생장정황화각농도조추수선감대OFs적억제솔.차외,분별재배양기중가입1×10-6、1×10-5、1×10-4 mol/L추수선감작용48 h,채용류식세포술급응용MCYCLE연건진행분석,용백분솔기록아이배체세포수거급세포주기각시기수거,검측각농도약물조급무약물배양기조OFs적조망솔화세포주기적분포；채용면역조직화학염색법측정전화생장인자-β(TGF-β)재OFs중적표체,이평개불동농도추수선감대OFs분비TGF-β공능적영향.결과 배양화전대적OFs정사형,파형단백면역화학염색위양성반응.1×10-4、1×10-5、1 ×10-6、1×10-7、1×10-8mol/L추수선감작용24、48화72 h,수착추수선감농도적증가,OFs적증생치(A450)축점하강,차이유통계학의의(F농도=62.004,P＜0.05)；차외,수착추수선감작용시간적증가,A450치축점하강,차이유통계학의의(F시간=459.582,P＜0.05)；수착추수선감농도적증가,기대OFs생장적억제솔축점승고.대조조、1×10-6、1×10-5、1×10-4 mol/L추수선감조OFs조망솔분별위(1.73±0.15)％、(21.04±4.56)％、(31.84±6.21)％화(35.32±5.56)％,4개조간차이유통계학의의(F=83.905,P＜0.05),각조간비교차이균유통계학의의(P＜0.05)；수착추수선감농도적증가,G2+M기세포백분수축점하강,차이유통계학의의(F=20.443,P＜0.05).면역조직화학법검측발현,재융합상태전적OFs가이분비TGF-β,양성솔위(97.60±2.09)％,이1×10-4 mol/L추수선감작용후OFs밀도명현감저,부분OFs양성솔위(44.43±3.96)％,차이유통계학의의(t=65.330,P＜0.05). 결론 추수선감가능통과감소OFs분비TGF-β억제TAO환자OFs적증생병유도OFs적조망,기작용정시간화제량의뢰성.
Background The orbital fibroblasts (OFs) in thyroid associated ophthalmopathy (TAO) play important roles in the proliferative and inflammatory response.Seeking the drug which inhibit OFs growth is of a vital significance for the prevention and treatment of TAO.Research documented that colchicine has an anti-fibrosis effect.But its influence on OFs of TAO patient is few known.Objective This study was to investigate the effect of colchicine on growth and apoptosis of OFs in vitro.Methods The retroobital connective tissue was obtained form 3 TAO patients and cultured using explant method.OFs were passaged and identified by immunochemistry,and 3-8 genetaions of cells were used in the study.Colchicine at the concentrations of 1 × 10-8,1 × 10-7,1 × 10-6,1 × 10-5,1 ×10-4 mol/L was added into the RPMI 1640 with 10％ fetal bovine serum(FBS) to incubated the cells for 24,48 and 72 hours respectively,and only RPMI 1640 was used to culture the cells as the control group.Cell counting kit-8 (CCK-8)was used to detect the absorbance value (A450) of OFs for the evaluatuion of OFs and the inhibitory rate of colchicine to OFs.The colchicine of 1 ×10-6,1 ×10-5,1 × 10-4 mol/L was added into the culture medium for 48 hours,and then the apoptotic rate of the cells and the cell percentage in various cellular cycle was assayed by flow cytometry(FCM).The expression of transforming growth factor-β (TGF-β)in the cells was detected by immunochemistry to assess the influence of colchicine on the serection of the cells.Results Cultured cells showed the spindle-like in shape and the cell number was significantly increased with the incubation time.After incubated with 1 × 10-4,1 × 10 5,1 × 10-6,1 ×10-7,1 × 10-8 mol/L colchicines,the A450 values were gradually reduced with the increase of the concentrations of colchicine(F ion =62.004,P＜0.05),and significant differences were found between different contrations of colchicine groups(all P＜0.05).Aslo,gradually declined A450 values of the cells were seen with the lapse of culture time among the groups(Ftime =459.582,P＜0.05).The inbitory rate of colchicine to the cells was elevated with the increase of concentrations.The apoptotic rates of the cells were (1.73 ± 0.15) ％,(21.04 ± 4.56) ％,(31.84 ±6.21)％and(35.32±5.56)％ in the control group and 1 × 10-6,1 × 10-5,1 × 10 4 mol/L colchicine groups respectively,with statistically significant difference among the 4 groups (F =83.905,P＜0.05).With the increase of concentrations of colchicines,the cell percentage in G2 +M phase lessened gradually,showing significant difference among the control group and the 1 × 10-6,1 × 10-5,1 × 10-4 mol/L colchicine groups (F =20.443,P＜0.05).The expression of the TGF-β in the cells was (97.60± 2.09) ％ in the control group,and that in the 1 × 10-4 mol/L colchicine group was (44.43 ± 3.96) ％,presenting a significant difference between them (t =65.330,P ＜ 0.05).Conclusions Colchicine can induce apoptosis of OFs and inhibit the prolilferation of OFs in a time-and dose-dependent manner probably by decreasing the TGF-β secretion
