中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
5期
428-433
,共6页
角膜肌成纤维细胞%细胞增生%多西环素
角膜肌成纖維細胞%細胞增生%多西環素
각막기성섬유세포%세포증생%다서배소
Corneal myofibroblast%Cell proliferation%Doxycycline
背景 多西环素是一种金属离子螯合剂和广谱抗生素,常用于眼表疾病的治疗. 目的 研究多西环素对体外培养的牛角膜肌成纤维细胞增生的影响,并探讨其作用机制.方法 收集6只新鲜牛角膜,分别用基础培养液配制的1.0g/L及2.0 g/L Ⅰ型胶原酶对牛角膜基质层进行二步法消化分离,制成细胞悬液后转入培养瓶中用RPMI-1640培养液(含质量分数10％胎牛血清)进行培养.取生长良好的细胞进行波形蛋白免疫细胞化学染色以确定所培养的细胞来自角膜基质层,在细胞传代培养的同时进行α-平滑肌肌动蛋白(α-SMA)免疫荧光细胞化学染色,呈阳性表现的细胞为角膜肌成纤维细胞.细胞培养36 h后培养液中不加入任何药物者为阴性对照组,阳性对照组细胞培养液中加入120 mg/L地塞米松,另分别在培养液中加入10、20、40、60、80 mg/L多西环素作为药物干预组.采用MTT比色法、流式细胞术测定各组干预24 h和48 h后角膜肌成纤维细胞的增生情况及细胞周期各时相的分布. 结果 体外分离培养的牛角膜肌成纤维细胞生长良好,免疫组织化学染色显示波形蛋白和α-SMA呈阳性反应,证实为角膜肌成纤维细胞.MTT比色法检测显示,随着多西环素质量浓度的增加,活性角膜肌成纤维细胞的数量逐渐下降,差异有统计学意义(F质量浓度=1233.778,P＜0.001)；随着药物作用时间的延长,活性角膜肌成纤维细胞的数量逐渐下降,差异有统计学意义(F时间=227.564,P＜0.001)；且两因素间的交互效应亦有统计学意义(F交互作用=51.656,P＜0.001).流式细胞术细胞周期分析显示,10、20、40、60、80 mg/L多西环素作用24 h,G0-G1期角膜肌成纤维细胞率分别为82.85％、84.36％、85.18％、87.12％、89.31％,明显高于阴性对照组的63.89％,差异均有统计学意义(P＜0.05),40 mg/L多西环素组G0-G1期角膜肌成纤维细胞率接近阳性对照组；10、20、40、60、80 mg/L多西环素作用48 h,G0-G1期角膜肌成纤维细胞率分别为82.78％、86.15％、88.23％、89.57％、93.00％,明显高于阴性对照组的70.17％,差异均有统计学意义(P＜0.01),而40 mg/L多西环素组G0-G1期角膜肌成纤维细胞百分数接近阳性对照组.结论 在10 ～ 80 mg/L质量浓度时,多西环素以剂量-时间效应依赖的方式抑制体外培养牛角膜肌成纤维细胞的增生,40 mg/L多西环素与120 mg/L地塞米松具有相当的作用效果.
揹景 多西環素是一種金屬離子螯閤劑和廣譜抗生素,常用于眼錶疾病的治療. 目的 研究多西環素對體外培養的牛角膜肌成纖維細胞增生的影響,併探討其作用機製.方法 收集6隻新鮮牛角膜,分彆用基礎培養液配製的1.0g/L及2.0 g/L Ⅰ型膠原酶對牛角膜基質層進行二步法消化分離,製成細胞懸液後轉入培養瓶中用RPMI-1640培養液(含質量分數10％胎牛血清)進行培養.取生長良好的細胞進行波形蛋白免疫細胞化學染色以確定所培養的細胞來自角膜基質層,在細胞傳代培養的同時進行α-平滑肌肌動蛋白(α-SMA)免疫熒光細胞化學染色,呈暘性錶現的細胞為角膜肌成纖維細胞.細胞培養36 h後培養液中不加入任何藥物者為陰性對照組,暘性對照組細胞培養液中加入120 mg/L地塞米鬆,另分彆在培養液中加入10、20、40、60、80 mg/L多西環素作為藥物榦預組.採用MTT比色法、流式細胞術測定各組榦預24 h和48 h後角膜肌成纖維細胞的增生情況及細胞週期各時相的分佈. 結果 體外分離培養的牛角膜肌成纖維細胞生長良好,免疫組織化學染色顯示波形蛋白和α-SMA呈暘性反應,證實為角膜肌成纖維細胞.MTT比色法檢測顯示,隨著多西環素質量濃度的增加,活性角膜肌成纖維細胞的數量逐漸下降,差異有統計學意義(F質量濃度=1233.778,P＜0.001)；隨著藥物作用時間的延長,活性角膜肌成纖維細胞的數量逐漸下降,差異有統計學意義(F時間=227.564,P＜0.001)；且兩因素間的交互效應亦有統計學意義(F交互作用=51.656,P＜0.001).流式細胞術細胞週期分析顯示,10、20、40、60、80 mg/L多西環素作用24 h,G0-G1期角膜肌成纖維細胞率分彆為82.85％、84.36％、85.18％、87.12％、89.31％,明顯高于陰性對照組的63.89％,差異均有統計學意義(P＜0.05),40 mg/L多西環素組G0-G1期角膜肌成纖維細胞率接近暘性對照組；10、20、40、60、80 mg/L多西環素作用48 h,G0-G1期角膜肌成纖維細胞率分彆為82.78％、86.15％、88.23％、89.57％、93.00％,明顯高于陰性對照組的70.17％,差異均有統計學意義(P＜0.01),而40 mg/L多西環素組G0-G1期角膜肌成纖維細胞百分數接近暘性對照組.結論 在10 ～ 80 mg/L質量濃度時,多西環素以劑量-時間效應依賴的方式抑製體外培養牛角膜肌成纖維細胞的增生,40 mg/L多西環素與120 mg/L地塞米鬆具有相噹的作用效果.
배경 다서배소시일충금속리자오합제화엄보항생소,상용우안표질병적치료. 목적 연구다서배소대체외배양적우각막기성섬유세포증생적영향,병탐토기작용궤제.방법 수집6지신선우각막,분별용기출배양액배제적1.0g/L급2.0 g/L Ⅰ형효원매대우각막기질층진행이보법소화분리,제성세포현액후전입배양병중용RPMI-1640배양액(함질량분수10％태우혈청)진행배양.취생장량호적세포진행파형단백면역세포화학염색이학정소배양적세포래자각막기질층,재세포전대배양적동시진행α-평활기기동단백(α-SMA)면역형광세포화학염색,정양성표현적세포위각막기성섬유세포.세포배양36 h후배양액중불가입임하약물자위음성대조조,양성대조조세포배양액중가입120 mg/L지새미송,령분별재배양액중가입10、20、40、60、80 mg/L다서배소작위약물간예조.채용MTT비색법、류식세포술측정각조간예24 h화48 h후각막기성섬유세포적증생정황급세포주기각시상적분포. 결과 체외분리배양적우각막기성섬유세포생장량호,면역조직화학염색현시파형단백화α-SMA정양성반응,증실위각막기성섬유세포.MTT비색법검측현시,수착다서배소질량농도적증가,활성각막기성섬유세포적수량축점하강,차이유통계학의의(F질량농도=1233.778,P＜0.001)；수착약물작용시간적연장,활성각막기성섬유세포적수량축점하강,차이유통계학의의(F시간=227.564,P＜0.001)；차량인소간적교호효응역유통계학의의(F교호작용=51.656,P＜0.001).류식세포술세포주기분석현시,10、20、40、60、80 mg/L다서배소작용24 h,G0-G1기각막기성섬유세포솔분별위82.85％、84.36％、85.18％、87.12％、89.31％,명현고우음성대조조적63.89％,차이균유통계학의의(P＜0.05),40 mg/L다서배소조G0-G1기각막기성섬유세포솔접근양성대조조；10、20、40、60、80 mg/L다서배소작용48 h,G0-G1기각막기성섬유세포솔분별위82.78％、86.15％、88.23％、89.57％、93.00％,명현고우음성대조조적70.17％,차이균유통계학의의(P＜0.01),이40 mg/L다서배소조G0-G1기각막기성섬유세포백분수접근양성대조조.결론 재10 ～ 80 mg/L질량농도시,다서배소이제량-시간효응의뢰적방식억제체외배양우각막기성섬유세포적증생,40 mg/L다서배소여120 mg/L지새미송구유상당적작용효과.
Background Doxycycline is a broad spectrum antibiotic,and it is frequently used in the treatment of ocular surface diseases.Objective The purpose of the present study was to investigate the effect of doxycycline on the inhibition of cell proliferation in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repairing mechanism.Methods Six fresh bovine corneas were collected.The corneal stromal layer was isolated by two-step method of 1.0 g/L and 2.0 g/L collegenase-1.Isolated cells were plated at mantaryay culture flask in 10％ FBS of RPMI-1640.Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry,and the cells with influoresccence staining for vimentin and α-SMA were identified as the corneal myofibroblasts.Doxycycline at the concentrations of 10,20,40,60,80 mg/L was added to the medium,respectively,in different concentrations of doxycycline groups.Dexamethasone (120 mg/L)was used in the same way in the positive control group,and no drug was used in the negative control group.Cell proliferation was evaluated by MTT and the cell cycle was analyzed by BD FACScan flow cytometer assay 24 hours and 48 hours after addition of any drug.Results The cells grew well and showed the positive response for vimentin and α-SMA.MTT assay showed that the A570values of bovine corneal myofibroblasts were gradually declined with the increase of the concentration of doxycycline and lapse of active time,showing statistically significant difference (Fconcentration =1233.778,P＜0.001 ; Ftime =227.564,P ＜ 0.001).And the difference between the two factors was also statistically significant (Ftime*concentration =51.656,P＜0.001).Flow cytometry cell cycle analysis showed that 24 hours after 10,20,40,60,80 mg/L doxycycline treated,the perentage of of corneal myofibroblast cell in G0-G1 phase was 82.85％,84.36％,85.18％,87.12 ％ and 89.31％,showing significant increase in comparison with 63.89％ of the negative control group (all P＜0.05),and that of 40 mg/L doxycycline group was near the positive control group.Forty-eight hours after 10,20,40,60,80 mg/L doxycycline treated,the perentage of of corneal myofibroblast cell in G0-G1 phase was 82.78％,86.15％,88.23％,89.57％,93.00％,with significant increase in comparison with 70.17％ of the negative control group (all P ＜ 0.01),and that of 40 mg/L doxycycline group was near the positive control group.Conclusions The growth of the bovine corneal myofibroblasts is inhibited by doxycycline in time-and dosedependent manner in the range from 10 mg/L to 80 mg/L,and 40 mg/L of doxycycline has an obviously inhibitory action as 120 mg/L dexamethasone.
