中华眼科杂志
中華眼科雜誌
중화안과잡지
Chinese Journal of Ophthalmology
2013年
5期
433-437
,共5页
刘曼丽%邹文进%黄明汉%赵静博%付馨余%王松
劉曼麗%鄒文進%黃明漢%趙靜博%付馨餘%王鬆
류만려%추문진%황명한%조정박%부형여%왕송
角膜%肌纤维母细胞%多西环素%核仁组成区%染色与标记%肌动蛋白类%细胞,培养的
角膜%肌纖維母細胞%多西環素%覈仁組成區%染色與標記%肌動蛋白類%細胞,培養的
각막%기섬유모세포%다서배소%핵인조성구%염색여표기%기동단백류%세포,배양적
Cornea%Myofibroblasts%Doxycycline%Nucleolus organizer regions%Staining and labeling%Actins%Cells,cultured
目的 探讨多西环素对角膜肌成纤维细胞核仁组成区嗜银蛋白(AgNOR)及α平滑肌肌动蛋白(α-SMA)表达的抑制作用.方法 实验研究.分别用基础培养液配制的1.0及2.0 g/LⅠ型胶原酶对牛角膜基质层进行二步消化分离,制成细胞悬液后转入培养瓶中用RPMI-1640培养液(含10％胎牛血清)培养.取生长良好的细胞进行波形蛋白免疫细胞化学染色和免疫荧光细胞化学染色.选用生长良好的牛角膜肌成纤维细胞分成3个组:阴性对照组(无药物干预),地塞米松组(地塞米松120mg/L)及各浓度梯度(10、20、40、60及80mg/L)多西环素组,每组30例.指标测定:采用AgNOR及α-SMA免疫荧光细胞化学染色等技术测定各实验组在对角膜肌成纤维细胞干预24和48h后,对细胞DNA复制及α-SMA合成的影响.各组间比较采用单因素方差分析,同一药物浓度下两组间比较采用两样本均数比较t检验.结果 在体外成功分离培养牛角膜肌成纤维细胞,显微镜下观察及α-SMA免疫荧光细胞化学染色后证实所培养的细胞为角膜肌成纤维细胞.细胞被干预24h后,阴性对照组AgNOR颗粒数为(6.40±0.62)个,AgNOR颗粒面积为(34.80±2.36) μm2；60mg/L多西环素组AgNOR颗粒数(2.23±0.43)个,AgNOR颗粒面积为(19.91 ±2.15) μm2.细胞被干预48h后,阴性对照组AgNOR颗粒数为(7.27±0.64)个,AgNOR颗粒面积为(36.27±1.99) μm2；60 mg/L多西环素组AgNOR颗粒数为(2.80±0.76)个,AgNOR颗粒面积为(13.75±2.09) μm2.差异有统计学意义(干预24 h:F=252.55,202.16;P ＜0.05;干预48 h:F=169.38,853.23;P ＜0.05;在同一浓度干预下:t=6.98,11.62;P ＜0.05).当多西环素浓度达60 mg/L时作用效果与120 mg/L地塞米松相当,差异无统计学意义(t=1.182,0.213;P ＞0.05).α-SMA免疫荧光细胞化学染色后行荧光定量分析,地塞米松对α-SMA的合成无明显作用(t=1.096,P =0.287);而多西环素组可抑制角膜肌成纤维细胞中α-SMA的合成,各浓度多西环素组细胞荧光染色的灰度值分别为189.90±7.48、140.20±7.79、113.20±8.98、98.00±3.50、85.50±4.99,差异有统计学意义(F=761.79,P=0.00).结论 在10 ～ 80 mg/L浓度范围内,多西环素可抑制体外培养的牛角膜肌成纤维细胞的AgNOR增生,呈剂量-时间-效应依赖性；在药物浓度达到60 mg/L时,与120 mg/L地塞米松拥有相当的作用效果.多西环素同时具有抑制细胞合成α-SMA的作用,与地塞米松比较,对抑制角膜瘢痕的形成更具研究价值.
目的 探討多西環素對角膜肌成纖維細胞覈仁組成區嗜銀蛋白(AgNOR)及α平滑肌肌動蛋白(α-SMA)錶達的抑製作用.方法 實驗研究.分彆用基礎培養液配製的1.0及2.0 g/LⅠ型膠原酶對牛角膜基質層進行二步消化分離,製成細胞懸液後轉入培養瓶中用RPMI-1640培養液(含10％胎牛血清)培養.取生長良好的細胞進行波形蛋白免疫細胞化學染色和免疫熒光細胞化學染色.選用生長良好的牛角膜肌成纖維細胞分成3箇組:陰性對照組(無藥物榦預),地塞米鬆組(地塞米鬆120mg/L)及各濃度梯度(10、20、40、60及80mg/L)多西環素組,每組30例.指標測定:採用AgNOR及α-SMA免疫熒光細胞化學染色等技術測定各實驗組在對角膜肌成纖維細胞榦預24和48h後,對細胞DNA複製及α-SMA閤成的影響.各組間比較採用單因素方差分析,同一藥物濃度下兩組間比較採用兩樣本均數比較t檢驗.結果 在體外成功分離培養牛角膜肌成纖維細胞,顯微鏡下觀察及α-SMA免疫熒光細胞化學染色後證實所培養的細胞為角膜肌成纖維細胞.細胞被榦預24h後,陰性對照組AgNOR顆粒數為(6.40±0.62)箇,AgNOR顆粒麵積為(34.80±2.36) μm2；60mg/L多西環素組AgNOR顆粒數(2.23±0.43)箇,AgNOR顆粒麵積為(19.91 ±2.15) μm2.細胞被榦預48h後,陰性對照組AgNOR顆粒數為(7.27±0.64)箇,AgNOR顆粒麵積為(36.27±1.99) μm2；60 mg/L多西環素組AgNOR顆粒數為(2.80±0.76)箇,AgNOR顆粒麵積為(13.75±2.09) μm2.差異有統計學意義(榦預24 h:F=252.55,202.16;P ＜0.05;榦預48 h:F=169.38,853.23;P ＜0.05;在同一濃度榦預下:t=6.98,11.62;P ＜0.05).噹多西環素濃度達60 mg/L時作用效果與120 mg/L地塞米鬆相噹,差異無統計學意義(t=1.182,0.213;P ＞0.05).α-SMA免疫熒光細胞化學染色後行熒光定量分析,地塞米鬆對α-SMA的閤成無明顯作用(t=1.096,P =0.287);而多西環素組可抑製角膜肌成纖維細胞中α-SMA的閤成,各濃度多西環素組細胞熒光染色的灰度值分彆為189.90±7.48、140.20±7.79、113.20±8.98、98.00±3.50、85.50±4.99,差異有統計學意義(F=761.79,P=0.00).結論 在10 ～ 80 mg/L濃度範圍內,多西環素可抑製體外培養的牛角膜肌成纖維細胞的AgNOR增生,呈劑量-時間-效應依賴性；在藥物濃度達到60 mg/L時,與120 mg/L地塞米鬆擁有相噹的作用效果.多西環素同時具有抑製細胞閤成α-SMA的作用,與地塞米鬆比較,對抑製角膜瘢痕的形成更具研究價值.
목적 탐토다서배소대각막기성섬유세포핵인조성구기은단백(AgNOR)급α평활기기동단백(α-SMA)표체적억제작용.방법 실험연구.분별용기출배양액배제적1.0급2.0 g/LⅠ형효원매대우각막기질층진행이보소화분리,제성세포현액후전입배양병중용RPMI-1640배양액(함10％태우혈청)배양.취생장량호적세포진행파형단백면역세포화학염색화면역형광세포화학염색.선용생장량호적우각막기성섬유세포분성3개조:음성대조조(무약물간예),지새미송조(지새미송120mg/L)급각농도제도(10、20、40、60급80mg/L)다서배소조,매조30례.지표측정:채용AgNOR급α-SMA면역형광세포화학염색등기술측정각실험조재대각막기성섬유세포간예24화48h후,대세포DNA복제급α-SMA합성적영향.각조간비교채용단인소방차분석,동일약물농도하량조간비교채용량양본균수비교t검험.결과 재체외성공분리배양우각막기성섬유세포,현미경하관찰급α-SMA면역형광세포화학염색후증실소배양적세포위각막기성섬유세포.세포피간예24h후,음성대조조AgNOR과립수위(6.40±0.62)개,AgNOR과립면적위(34.80±2.36) μm2；60mg/L다서배소조AgNOR과립수(2.23±0.43)개,AgNOR과립면적위(19.91 ±2.15) μm2.세포피간예48h후,음성대조조AgNOR과립수위(7.27±0.64)개,AgNOR과립면적위(36.27±1.99) μm2；60 mg/L다서배소조AgNOR과립수위(2.80±0.76)개,AgNOR과립면적위(13.75±2.09) μm2.차이유통계학의의(간예24 h:F=252.55,202.16;P ＜0.05;간예48 h:F=169.38,853.23;P ＜0.05;재동일농도간예하:t=6.98,11.62;P ＜0.05).당다서배소농도체60 mg/L시작용효과여120 mg/L지새미송상당,차이무통계학의의(t=1.182,0.213;P ＞0.05).α-SMA면역형광세포화학염색후행형광정량분석,지새미송대α-SMA적합성무명현작용(t=1.096,P =0.287);이다서배소조가억제각막기성섬유세포중α-SMA적합성,각농도다서배소조세포형광염색적회도치분별위189.90±7.48、140.20±7.79、113.20±8.98、98.00±3.50、85.50±4.99,차이유통계학의의(F=761.79,P=0.00).결론 재10 ～ 80 mg/L농도범위내,다서배소가억제체외배양적우각막기성섬유세포적AgNOR증생,정제량-시간-효응의뢰성；재약물농도체도60 mg/L시,여120 mg/L지새미송옹유상당적작용효과.다서배소동시구유억제세포합성α-SMA적작용,여지새미송비교,대억제각막반흔적형성경구연구개치.
Objective To investigate the effect of doxycycline on the nucleolar organizing regions and a-smooth muscle actin expression in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repair mechanisms.Methods Cell culture and identification:bovine corneal fibroblasts were cultured after the stroma was incubated in 1.0 and 2.0 g/L type Ⅰ collagenase in two stages.Isolated cells were plated at mantaryay culture flask in 10％ of BSA RPMI-1640.Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry.The cells staining positive for Vimentin and α-SMA indicated the presence of corneal myofibroblasts.Bovine corneal myofibroblasts were treated with different concentrations of doxycycline (10,20,40,60,80 mg/L),a bland control group and the dexamethasone group (120 mg/L) were set up,each group had 30 cases.The argyrophilic nucleolar organizing regions (AgNOR) staining and the immunohistochemistry for α-SMA were performed when the cells were treated for 24 hours and 48 hours.The AgNOR count (Ag-c),AgNOR area (Ag-a) and the expression of α-SMA in the bovine corneal myofibroblasts among each experiment group and control group were compared using one-way ANOVA,further pairwise comparisons using Independent-Samples t test.Results Cell culture techniques were successfully used to establish a method for the isolation and culture of bovine corneal myofibroblasts.Microscopic examination and immunohistochemical staining confirmed that the cells cultured were bovine corneal myofibroblasts.The Ag-c and Ag-a of bovine corneal myofibroblasts progressively decreased as the concentrations of doxycycline was increase.24 h:bland control group Ag-c was 6.40 ± 0.6,60 mg/L doxycycline group Ag-c was 2.23 ± 0.43;bland control group Ag-a was (34.80 ± 2.36) μm2,60 mg/L doxycycline hormone group Ag-a was (19.91 ± 2.15) μm2.48 h:bland control group Ag-c was 7.27 ± 0.6,60 mg/L doxycycline hormone group Ag-c was 2.80 ± 0.76,bland 2control group Ag-a was (36.27 ± 1.99) μ m2,60 mg/L doxycycline group Ag-a was (13.75 ± 2.09) μm2.The differences were statistically significant:in the same time intervention (FAg-c 24 h =252.55,FAg-a24 h =202.16,P ＜ 0.05,FAg-c48h =169.38,FAg-a48h =853.23,P ＜0.05),in the same concentrations intervention (tAg-e =6.98,tAg-a =11.62,P ＜ 0.05).And 60 mg/L of doxycycline had an obviously inhibitory action as 120 mg/L dexamethasone in the same treated hours (dexamethasone group Ag-a 24 h =30.56 ± 3.66,dexamethasone group Ag-a 48 h =28.35 ± 1.23),the differences were not statistically significant (tAg-a 24h =1.182,P =0.242,tAg-a 48 h =0.21,P =0.832).As the concentrations investigated,doxycycline can inhibit the expression of α-SMA in the bovine corneal myofibroblasts (189.90 ± 7.48,140.20 ± 7.79,113.20 ± 8.98,98.00 ± 3.50,85.50 ± 4.99),the difference was statistically significant (F =761.79,P =0.00).While dexamethasone had no significant role in the expression of α-SMA (bland control group was 225.10 ± 6.74,the dexamethasone group was 228.50 ± 7.12),and the statistically difference was not obvious(t =1.096,P =0.287).Conclusions As the concentrations of doxycycline was increased from 10 mg/L to 80 mg/L,the AgNOR count and AgNOR area of bovine corneal myofibroblasts can be significantly reduced in vitro.Compared with dexamethasone,doxycycline significantly suppressed the expression of α-SMA in bovine corneal myofibroblasts in a dose-dependent positive trend.(Chin J Ophthalmol,2013,49:)
