中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
2期
119-124
,共6页
陈宁虹%毕宏生%郭大东%郭媛媛%杜然然%马晓华
陳寧虹%畢宏生%郭大東%郭媛媛%杜然然%馬曉華
진저홍%필굉생%곽대동%곽원원%두연연%마효화
紫杉醇/药理学%青光眼/手术%Tenon囊/细胞学%成纤维细胞/细胞学%基质金属蛋白酶-1
紫杉醇/藥理學%青光眼/手術%Tenon囊/細胞學%成纖維細胞/細胞學%基質金屬蛋白酶-1
자삼순/약이학%청광안/수술%Tenon낭/세포학%성섬유세포/세포학%기질금속단백매-1
Paclitaxe/pharmacology%Glaucoma/surgery%Tenon capsule/ cytology%Fibroblast/cytology%Matrix metalloproteinase-1
背景 人Tenon囊成纤维细胞(HTFs)的异常增生是导致抗青光眼滤过手术失败的主要原因.寻找抑制HTFs增生的药物对抑制青光眼术后功能性滤过泡的瘢痕化有重要意义. 目的 观察紫杉醇对体外培养的HTFs增生、凋亡、细胞周期以及基质金属蛋白酶-1(MMP-1)表达的影响.方法 收集抗青光眼滤过术中切除的人眼Tenon囊组织,用组织块培养法培养HTFs并进行传代,取3～6代细胞用于实验.采用小鼠抗人角蛋白抗体、小鼠抗人波形蛋白抗体、小鼠抗人结蛋白抗体、小鼠抗人S-100抗体行免疫组织化学染色鉴定培养的HTFs.在培养基中分别加入0、1×10-8、1 × 10-7、1 × 10-6 mol/L紫杉醇,采用实时细胞电子分析系统(RT-CES)观察不同浓度紫杉醇作用后HTFs的细胞指数变化；采用DAPI染色法观察各组细胞的凋亡情况；用流式细胞术检测不同浓度紫杉醇作用后HTFs各细胞周期比例；采用实时定量PCR(real-time PCR)和ELISA法检测各组HTFs中MMP-1 mRNA和蛋白的表达量,分别以MMP-1 mRNA/GAPDH mRNA和MMP-1蛋白质量浓度(μg/L)表示.结果 组织块原代培养7d内可见细胞游出,呈长梭形和不规则三角形,培养的细胞抗波形蛋白抗体染色阳性,而抗角蛋白抗体、抗结蛋白抗体和抗S-100抗体染色阴性.培养基中加入紫杉醇作用24 h后,1×10-8、1×10-7、1×10-6 mol/L紫杉醇组平均细胞指数值分别为1.093±0.191、0.665±0.093和0.473±0.117,均明显低于0 mol/L紫杉醇组的1.514±0.283,差异均有统计学意义(均P=0.000)；紫杉醇作用后,HTFs的细胞核内可见呈DAPI蓝色荧光的颗粒状物质,荧光强度随着紫杉醇浓度的增加而增强.1×10-8、1×10-7、1×10-6 moL/L紫杉醇分别作用12h后,G2/M期HTFs的比例分别为(9.20±0.80)％、(12.37±0.45)％和(13.80±0.35)％,明显高于0 mol/L紫杉醇组的(7.17±0.50)％,差异均有统计学意义(P=0.005、0.000、0.000).与0 mol/L紫杉醇组比较,1×10-8、1 ×10-7、1 ×10-6 mol/L紫杉醇作用30 min后HTFs中MMP-1mRNA的相对表达量均明显下降,差异均有统计学意义(均P=0.000)；1×10-8、1×10.、1×106mol/L紫杉醇作用24 h后,HTFs中MMP-1蛋白的表达量均明显低于0 mol/L紫杉醇组,差异均有统计学意义(P=0.010、0.002、0.001).结论 1×10-8 ～ 1×10-6 mol/L紫杉醇可抑制体外培养HTFs的增生,诱导细胞凋亡,阻断细胞的有丝分裂,其作用机制可能与下调细胞中MMP-1的表达有关.
揹景 人Tenon囊成纖維細胞(HTFs)的異常增生是導緻抗青光眼濾過手術失敗的主要原因.尋找抑製HTFs增生的藥物對抑製青光眼術後功能性濾過泡的瘢痕化有重要意義. 目的 觀察紫杉醇對體外培養的HTFs增生、凋亡、細胞週期以及基質金屬蛋白酶-1(MMP-1)錶達的影響.方法 收集抗青光眼濾過術中切除的人眼Tenon囊組織,用組織塊培養法培養HTFs併進行傳代,取3～6代細胞用于實驗.採用小鼠抗人角蛋白抗體、小鼠抗人波形蛋白抗體、小鼠抗人結蛋白抗體、小鼠抗人S-100抗體行免疫組織化學染色鑒定培養的HTFs.在培養基中分彆加入0、1×10-8、1 × 10-7、1 × 10-6 mol/L紫杉醇,採用實時細胞電子分析繫統(RT-CES)觀察不同濃度紫杉醇作用後HTFs的細胞指數變化；採用DAPI染色法觀察各組細胞的凋亡情況；用流式細胞術檢測不同濃度紫杉醇作用後HTFs各細胞週期比例；採用實時定量PCR(real-time PCR)和ELISA法檢測各組HTFs中MMP-1 mRNA和蛋白的錶達量,分彆以MMP-1 mRNA/GAPDH mRNA和MMP-1蛋白質量濃度(μg/L)錶示.結果 組織塊原代培養7d內可見細胞遊齣,呈長梭形和不規則三角形,培養的細胞抗波形蛋白抗體染色暘性,而抗角蛋白抗體、抗結蛋白抗體和抗S-100抗體染色陰性.培養基中加入紫杉醇作用24 h後,1×10-8、1×10-7、1×10-6 mol/L紫杉醇組平均細胞指數值分彆為1.093±0.191、0.665±0.093和0.473±0.117,均明顯低于0 mol/L紫杉醇組的1.514±0.283,差異均有統計學意義(均P=0.000)；紫杉醇作用後,HTFs的細胞覈內可見呈DAPI藍色熒光的顆粒狀物質,熒光彊度隨著紫杉醇濃度的增加而增彊.1×10-8、1×10-7、1×10-6 moL/L紫杉醇分彆作用12h後,G2/M期HTFs的比例分彆為(9.20±0.80)％、(12.37±0.45)％和(13.80±0.35)％,明顯高于0 mol/L紫杉醇組的(7.17±0.50)％,差異均有統計學意義(P=0.005、0.000、0.000).與0 mol/L紫杉醇組比較,1×10-8、1 ×10-7、1 ×10-6 mol/L紫杉醇作用30 min後HTFs中MMP-1mRNA的相對錶達量均明顯下降,差異均有統計學意義(均P=0.000)；1×10-8、1×10.、1×106mol/L紫杉醇作用24 h後,HTFs中MMP-1蛋白的錶達量均明顯低于0 mol/L紫杉醇組,差異均有統計學意義(P=0.010、0.002、0.001).結論 1×10-8 ～ 1×10-6 mol/L紫杉醇可抑製體外培養HTFs的增生,誘導細胞凋亡,阻斷細胞的有絲分裂,其作用機製可能與下調細胞中MMP-1的錶達有關.
배경 인Tenon낭성섬유세포(HTFs)적이상증생시도치항청광안려과수술실패적주요원인.심조억제HTFs증생적약물대억제청광안술후공능성려과포적반흔화유중요의의. 목적 관찰자삼순대체외배양적HTFs증생、조망、세포주기이급기질금속단백매-1(MMP-1)표체적영향.방법 수집항청광안려과술중절제적인안Tenon낭조직,용조직괴배양법배양HTFs병진행전대,취3～6대세포용우실험.채용소서항인각단백항체、소서항인파형단백항체、소서항인결단백항체、소서항인S-100항체행면역조직화학염색감정배양적HTFs.재배양기중분별가입0、1×10-8、1 × 10-7、1 × 10-6 mol/L자삼순,채용실시세포전자분석계통(RT-CES)관찰불동농도자삼순작용후HTFs적세포지수변화；채용DAPI염색법관찰각조세포적조망정황；용류식세포술검측불동농도자삼순작용후HTFs각세포주기비례；채용실시정량PCR(real-time PCR)화ELISA법검측각조HTFs중MMP-1 mRNA화단백적표체량,분별이MMP-1 mRNA/GAPDH mRNA화MMP-1단백질량농도(μg/L)표시.결과 조직괴원대배양7d내가견세포유출,정장사형화불규칙삼각형,배양적세포항파형단백항체염색양성,이항각단백항체、항결단백항체화항S-100항체염색음성.배양기중가입자삼순작용24 h후,1×10-8、1×10-7、1×10-6 mol/L자삼순조평균세포지수치분별위1.093±0.191、0.665±0.093화0.473±0.117,균명현저우0 mol/L자삼순조적1.514±0.283,차이균유통계학의의(균P=0.000)；자삼순작용후,HTFs적세포핵내가견정DAPI람색형광적과립상물질,형광강도수착자삼순농도적증가이증강.1×10-8、1×10-7、1×10-6 moL/L자삼순분별작용12h후,G2/M기HTFs적비례분별위(9.20±0.80)％、(12.37±0.45)％화(13.80±0.35)％,명현고우0 mol/L자삼순조적(7.17±0.50)％,차이균유통계학의의(P=0.005、0.000、0.000).여0 mol/L자삼순조비교,1×10-8、1 ×10-7、1 ×10-6 mol/L자삼순작용30 min후HTFs중MMP-1mRNA적상대표체량균명현하강,차이균유통계학의의(균P=0.000)；1×10-8、1×10.、1×106mol/L자삼순작용24 h후,HTFs중MMP-1단백적표체량균명현저우0 mol/L자삼순조,차이균유통계학의의(P=0.010、0.002、0.001).결론 1×10-8 ～ 1×10-6 mol/L자삼순가억제체외배양HTFs적증생,유도세포조망,조단세포적유사분렬,기작용궤제가능여하조세포중MMP-1적표체유관.
Background The excessive growth of human Tenon fibroblasts (HTFs) is a primary cause of failure of anti-glaucomatous filtering surgery.To seek a drug of anti-fibrosis is of an important significance for improving the successful rate of anti-glaucomatous filtration surgery.Objective The goal of this study was to investigate the effect of paclitaxel on proliferation of HTFs in vitro.Methods Human Tenon tissue was obtained during the anti-glaucomatous filtering surgery.HTFs were cultivated using explant method and 3-6 generations of cells were used in the experiment.The cells were identified by immunochemistry using keratin,vimentin,fibronectin and S-100.Different concentrations (0,1 ×10-s,1 × 10-7,1 × 10-6 mol/L) of paclitaxel were added into the medium,and then the cell indexes (CI) in the various groups were detected by real-time cell electronic sensing (RT-CES) 24 hours after affection of paclitaxel.Apoptosis of the cells was examined using DAPI staining,and the proportion of the cells in different cycles were assayed by flow cytorneter 12 hours after addition of paclitaxel.The expressions of matrix metalloproteinase-1 (MMP-1) protein and mRNA were detected by ELISA and real-time PCR,respectively.Results The cells migrated from the cultivated tissue within 7 days with the fibrocyte-like shape.The cells showed the positive response for vimentin and absent response for keratin,fibronectin and S-100.The CI values were 1.093 ±0.191,0.665 ± 0.093 and 0.473 ± 0.117 in the 1 × 10-8,1 × 10-7 and 1 × 10-6 mol/L paclitaxel groups,showing significant rise in comparison with the 1.514 ±0.283 of the 0 mol/L paclitaxel group (all at P =0.000).The cell nuclei were normal in the 0 mol/L paclitaxel group,however,blue-fluorescent particles and apoptotic bodied were found in the cell nuclei after affection of paclitaxel.The proportion of G2/M phase of cells were (9.20±0.80) ％,(12.37±0.45)％ and (13.80±0.35)％ in the 1×10-8 mol/L,1×10-7 mol/L and 1×10-6 mol/L paclitaxel groups,which were higher than the (7.17±0.50) ％ in the 0 mol/L paclitaxel group (P=0.005,0.000,0.000).In addition,the relative expressing level of M MP-1 mRNA (MMP-1 mRNA/GAPDH mRNA) and the expression level of MMP-1 protein in the HTFs were significantly lower in the 1 ×10-8 mol/L,1 × 10-7 mol/L and 1 × 10-6 mol/L paclitaxel groups than those in the 0 mol/L group (all at P＜0.05).Conclusions Paclitaxel at the concentrations of 1 × 10-8 mol/L-1 × 10-6 mol/L inhibits the proliferation of HTFs in vitro by arresting the cellular mitosis and inducing cell apoptosis.These effects probably associated with down-regulation of MMP-1 expression in the HTFs.
