苯扎氯铵对人结膜上皮细胞毒性的研究

目的 探讨苯扎氯铵(BAC)对无血清培养法培养的人结膜上皮细胞的细胞毒性.方法 采用无血清培养的3-5代人结膜上皮细胞,将不同浓度的BAC(O.01%,0.005%,0.001%,0.0005%,0.0001%)单次作用于结膜上皮细胞15min,分别于处理细胞后6,12,24,48hMTT法检测细胞活力,于处理细胞后48h固定,扫描电镜观察拍照.结果 自BAC处理后6h起,与对照组相比,各浓度BAC均使细胞活力下降(P＜0.01).经过48小时的恢复期,细胞活力未见恢复.随BAC浓度增高,细胞表面微绒毛消失,细胞出现不同程度收缩至脱落至大部分溶解坏死.结论 BAC对于培养的人结膜上皮细胞具有细胞毒性,其毒性作用呈剂量依赖性.     

苯扎氯胺对体外培养的人角膜上皮细胞毒性作用的研究

目的 观察滴眼液中最常用的防腐剂-苯扎氯胺(BAC)对体外培养的人角膜上皮细胞的毒性作用.方法 建立人角膜上皮细胞的体外培养模型.不同质量浓度的BAC(0.1%、0.01%、0.001%、0.0001%)与细胞共同孵育,时间分别为10min、30min,2、6、24、48h.评估细胞毒性的方法 :MTT分析,光镜和扫描电镜观察细胞形态学改变.结果 0.1?C导致细胞立即死亡,MTT值在暴露时间为10min时显著下降(P＜0.01),30min降至最低(P＜0.01);光镜下细胞迅速脱壁.0.01?C在24h内导致细胞缓慢死亡,MTT值逐渐下降;扫描电镜示细胞的形态学改变为表面微绒毛脱落,细胞膜破损;0.001%～0.0001?C对细胞的损害出现在24h后.结论 BAC对体外培养的人角膜上皮细胞具有明确的毒性作用,即使在滴眼液中含有的常规质量浓度(0.01%)及低于常规质量浓度下仍可出现.部分临床长期使用含有BAC滴眼液患者可出现眼表问题.     

结膜松弛症结膜组织及泪液中黏蛋白的表达变化

目的:观察结膜松弛症松弛结膜组织和泪液中黏蛋白的表达变化,探讨结膜松弛症的发病机制.方法:收集结膜松弛症组38例38眼和正常对照组36例36眼的结膜组织和泪液标本,分别行免疫组织化学染色检测结膜组织中黏蛋白(MUC2、MUC4、MUC5AC、MUC16)表达情况,ELISA检测泪液中黏蛋白(MUC2、MUC4、MUC5AC、MUC16)A值,结果行统计学分析,比较两组之间的差异.结果:结膜松弛症组的结膜上皮细胞中MUC2、MUC4与正常对照组表达无统计学差异 (P=0.315、0.156);结膜松弛症组的结膜上皮细胞中MUC5AC、MUC16阳性表达细胞较正常对照组明显减少,差异有统计学意义(P=0.016、<0.01).结膜松弛症组泪液中MUC2的A值与正常对照组无统计学差异(P=0.651),结膜松弛症组中MUC4、MUC5AC的A值较正常对照组明显降低,有显著的统计学差异(均P<0.01),结膜松弛症组中MUC16的A值较正常对照组下降,差异有统计学意义(P=0.022).结论:结膜松弛症患者结膜组织和泪液中MUC5AC和MUC16均下降,对其进一步研究可能揭示结膜松弛症的发病机制.     

几丁聚糖对兔结膜上皮细胞和结膜成纤维细胞增殖的影响

目的　研究几丁聚糖对兔结膜上皮细胞和结膜成纤维细胞增殖的影响。方法　采用消化法培养结膜上皮细胞 ,组织块法培养结膜成纤维细胞。通过MTT比色法和细胞计数法测定几丁聚糖对两种细胞增殖的影响。结果　几丁聚糖在浓度 >0 0 3 %时能促进结膜上皮细胞的增殖 ,浓度为 0 12 %时促进作用最明显。结膜成纤维细胞在几丁聚糖浓度 >0 0 3 %时其增殖即受到抑制 ,抑制作用呈浓度依赖性。结论　几丁聚糖有望用于预防睑球粘连     

γ-干扰素对培养人结膜上皮细胞及泪腺上皮细胞的影响

γ-干抗素(interferon-γ,IFN-γ)是致炎作用很强的细胞因子,在机体各类炎性反应过程中起着重要的作用，它不仅可以诱导多种上皮缅胞过量表达细胞间黏附分子-1（intercellular adbesion molecule-1,ICAM-1,CD54)、主要组织相容性抗原(HLA-DR)等，而且可诱导细胞凋亡。有研究发现，Sjogren患者泪腺及涎腺中IFN-γ、白细胞介素-1(IL-1)及CD54的表达增高，但其相互关系，尤其IFN-γ人结膜和泪腺上皮细胞的炎性损伤作用尚不十分清楚。     

人结膜上皮细胞的培养鉴定及液氮冻存

目的：探索人眼结膜上皮细胞体外培养和细胞保存的最佳方法,建立人结膜上皮细胞,进一步研究人结膜的病理、生理特点及为毒理试验提供可靠的细胞模型。方法：分别用组织块培养法、机械分离法及混合消化液培养法体外培养正常成年人结膜上皮细胞,通过观察细胞形态、生长特性并用原位免疫组化方法鉴定培养细胞;收集第３代和第４代融合的细胞液氮冻存,保存３０天后复苏,观察复苏成功率。结果：３种取材方法中,混合消化液培养法细胞     

不含苯扎氯铵的抗青光眼滴眼液疗效及安全性的Meta分析

目的通过荟萃分析评价不含苯扎氯铵(BAK)的滴眼液治疗青光眼和高眼压症的有效性及安全性。设计荟萃分析。研究对象Medline(1966-2011年)、EMbase(1966-2010年)、Cochrane图书馆(2010年)及中国生物医学文献数据库CBM(1979-2010年)有关含与不含BAK的滴眼液治疗青光眼和高眼压症的临床对照研究文献资料。方法采用Cochrane系统评价的方法检索文献,按照纳入和排除标准限定研究对象,通过Jadad评分量表进行文献质量评估后,使用Cochrane协作网提供的RevMan 4.2统计软件进行Meta分析,以获得两者治疗青光眼和高眼压症的疗效及安全性是否有差异的相关证据。主要指标眼压,药物不良反应。结果纳入含与不含BAK的滴眼液治疗青光眼和高眼压症的临床对照研究6项(2313眼)。Meta分析结果显示,两类滴眼液均能有效降低眼压,两类滴眼液的降眼压幅度差异为0.08 mm Hg(95%CI,-0.10~0.27)(P=0.38)。随访期内发生相对较多的三种药物不良反应为结膜充血(10.98%)、过敏性结膜炎(4.84%)和干眼(3.11%)。采用固定效应模型进行Mete分析,用两类滴眼液者发生结膜充血、过敏性结膜炎和眼干的合并比值比分别为1.37(95%CI,1.05~1.80)、2.1(95%CI,1.26~3.48)和1.69(95%CI,1.13~2.53)(P<0.05)。结论不含BAK的滴眼液降眼压效果与含有BAK的滴眼液的差异无统计学意义且安全性更高,但尚需更多高质量的前瞻性临床对照研究进一步证实。     

rhEGF促进人结膜上皮细胞增殖的实验研究

目的：探讨rnEGF（recombinant buman epidermal growth factor)促进培养人结膜上皮细胞增殖的作用及最佳有效浓度,为提高眼表重建术后成功率寻找有效的治疗手段。方法：在传代人结膜上皮细胞中分别加入1μg/ml-100μg/ml浓度的rhEGF,于加药后第24、48、72、96h分别采用MTT法得出光吸收值以测定活性细胞数。结果：24hEGF各浓度组与对照组光吸收值无明显差异。48h50μg/ml、20μg/ml、10μg/mlEGF组光吸收值明显高于对照组,72h除100μg/ml组与其余EGF浓度组有显著性差异。结论：EGF对结膜上皮的增殖有明显促进作用,浓度为10－50μg/ml可以获得较好的效果。     

“去上皮”羊膜对体外培养的结膜上皮细胞的作用

目的：探讨羊膜基底膜对结膜上皮细胞形态功能的影响。方法：把“去上皮”羊膜按照基底膜或实质面向上分为两组,接种结膜上皮细胞,同样条件下培养,取培养14天的两组标本进行组织学（HE和PAS染色）,透射电镜,以及免疫组化检查（包括抗角蛋白和抗波形蛋白染色）。结果：细胞72小时内贴附在羊膜上,基底膜面接种的上皮细胞顶端见微绒毛,单层排列,细胞间有丰富桥粒,基底膜完整,可见半桥粒,抗角蛋白染色阳性,抗波形蛋白染色阴性;实质面接种的上皮细胞浆内有大量脂滴,表面未见微绒毛,细胞松散堆积,与羊膜连接不紧密,未见桥粒和半桥粒,抗角蛋白染色弱阳性,抗波形蛋白染色强阴性。结论：羊膜基底膜是结膜上皮细胞体外培养的良好载体,有利于上皮细胞生长,黏附,分化,并能减缓上皮细胞老化。     

多西环素对培养的人结膜上皮细胞炎性反应及凋亡的影响

目的探讨多西环素对体外培养的人结膜上皮细胞细胞黏附分子1（ICAM-1,CD54）、人类白细胞抗原DR（HLA—DR）、白细胞介素1β（IL-1β）表达及凋亡的影响。方法混合消化液培养法培养人结膜上皮细胞,免疫组织化学方法进行细胞鉴定。培养至第3代或第4代,在细胞培养液中分别加入γ干扰素（IFN-γ）0U／ml、IFN-γ300U／ml、IFN-γ300U／ml＋多西环素10μg／ml、IFN-γ300U／ml＋多西环素20μg／ml、IFN-γ300U／ml＋多西环素40μg／ml、IFN-γ300U／ml＋地塞米松100μg／ml,24h后收集细胞,流式细胞学检测CD54、HLA—DR及IL-1β的表达,Western blot检测CD54的表达。72h后收集细胞,碘化丙啶（PI）染色,流式细胞学检测凋亡。结果培养的正常人结膜上皮细胞可以表达少许CD54和IL-1β,加入IFN-γ后表达明显增加,加入多西环素和地塞米松后CD54和IL-1β的表达下降（P〈0．01）。随着所加多西环素浓度的增加,CD54和IL-1β的表达逐渐降低（P〈0．01）。培养的正常人结膜上皮细胞未发现明显的HLA—DR的表达及凋亡的产生,加入IFN-γ及多西环素后HLA—DR的表达及凋亡水平无明显变化（P〉0．05）。结论多西环素可抑制培养的正常人结膜上皮细胞CD54和IL-1β等炎性相关因子的产生,提示多西环素对干眼等非感染性眼表炎性疾病的治疗可能具有一定的应用前景。（中华眼科杂志,2005,41：842-846）     

"去上皮"羊膜对体外培养结膜上皮细胞的作用

目的 :探索羊膜基底膜对结膜上皮细胞形态功能的影响。方法 :把“去上皮”羊膜按照基底膜或实质面向上分为两组 ,接种结膜上皮细胞 ,同样条件下培养。取培养 14天的两组标本进行组织学 (HE和PAS染色 ) ,透射电镜 ,以及免疫组化检查 (包括抗角蛋白和抗波形蛋白染色 )。结果 :细胞 72小时内贴附在羊膜上。基底膜面接种的上皮细胞顶端见微绒毛 ,单层排列 ,细胞间有丰富桥粒 ,基底膜完整 ,可见半桥粒 ,抗角蛋白染色阳性 ,抗波形蛋白染色阴性 ;实质面接种的上皮细胞胞浆内有大量脂滴 ,表面未见微绒毛 ,细胞松散堆积 ,与羊膜连接不紧密 ,未见桥粒和半桥粒 ,抗角蛋白染色弱阳性 ,抗波形蛋白染色强阳性。结论 :羊膜基底膜是结膜上皮细胞体外培养的良好载体 ,有利于上皮细胞生长、粘附、分化 ,并能减缓上皮细胞老化。     

Effects of benzalkonium chloride on growth and survival of Chang conjunctival cells

PURPOSE: The aim of this study was to investigate the action of benzalkonium chloride (BAC), used as a preservative in most ophthalmic topical solutions, on epithelial conjunctival cells in vitro. METHODS: A continuous human conjunctival cell line (Wong-Kilbourne derivative of Chang conjunctiva) was exposed to BAC solutions at various concentrations (0.1%-0.0001%) during a period of 10 minutes. Cells were examined before treatment and 3, 24, 48, and 72 hours later, after reexposure to normal cell culture conditions. Cell number and viability were assessed with crystal violet and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide colorimetric assays. The expression of the apoptotic marker Apo 2.7, nuclear antigen p53, membrane proteins Fas and Fas ligand, and DNA content was studied by flow cytometry. Morphologic aspects of cell nuclei were analyzed on slides with a nucleic acid-specific dye, 4',6'-diamidino-2-phenylindole dihydrochloride. Cytoskeleton was labeled with a monoclonal anti-pancytokeratin antibody. In addition, apoptosis was measured by DNA electrophoresis assays in agarose gel. RESULTS: Cell exposure to 0.1% and 0.05% BAC induced cell lysis immediately after treatment. All cells (100%) treated with 0.01% BAC died in a delayed manner within 24 hours, with most of the characteristics of apoptosis (chromatin condensation and DNA fragmentation, reduction in cell volume, expression of the apoptotic marker Apo 2.7, and apoptotic changes in DNA content). Aliquots of 0.005%, 0.001%, 0.0005%, and 0.0001% BAC induced growth arrest and apoptotic cell death in a dose-dependent manner between 24 and 72 hours after treatment. The expressions of Fas and p53 did not vary after BAC treatment. Fas ligand was always negative. CONCLUSIONS: These results suggest that BAC induces cell growth arrest and death at a concentration as low as 0.0001%. The mode of BAC-induced cell death is dose-dependent. Cells die by necrosis after BAC treatment at high concentrations and by apoptosis if low concentrations of BAC are applied. This new aspect of in vitro toxicity of BAC could in part explain some ocular surface disorders observed in patients undergoing long-term topical treatments with preservative-containing drugs.     

"去上-皮"羊膜對體外培養的結膜上皮細胞的作用

目的探索羊膜基底膜對結膜上皮細胞形態功能的影響.方法把"去上皮"羊膜按照基底膜或實質面向上分為兩組,接種結膜上皮細胞,同樣條件下培養.取培養14天的兩組標本進行組織學(HE和PAS染色),透射電鏡,以及免疫組化檢查(包括抗角蛋白和抗波形蛋白染色).結果細胞72小時内貼附在羊膜上.基底膜面接種的上皮細胞頂端見微絨毛,單層排列,細胞間有豐富橋粒,基底膜完整,可見半橋粒,抗角蛋白染色陽性,抗波形蛋白染色陰性;實質面接種的上皮細胞漿内有大量脂滴,表面未見微絨毛,細胞鬆散堆積,與羊膜連接不緊密,未見橋粒和半橋粒,抗角蛋白染色弱陽性,抗波形蛋白染色强陽性.結論羊膜基底膜是結膜上皮細胞體外培養的良好載體,有利于上皮細胞生長、黏附、分化,并能减緩上皮細胞老化.     

外源性细胞周期蛋白激酶抑制因子p21基因对人晶状体上皮细胞周期的影响

目的 研究细胞周期蛋白激酶抑制因子p21基因转染对人晶状体上皮细胞(1ens epithelial cells,LEC)周期调控的影响,在基因水平探讨防治后发性白内障的可能性。方法 构建含人p21基因真核表达的载体质粒pcDNA3／p21,采用基因转染技术将质粒DNA转染至永生性人LEC系HLE-B3,使用流式细胞仪观察细胞生长和周期的变化,并采用逆转录聚合酶链式反应方法检测p21mRNA的表达;分别采用免疫组化和免疫印迹(western blotting,WB)方法检测p21蛋白的表达。结果 体外构建的载体质粒pcDNA3／p21经酶切鉴定含有人p21基因全长cDNA片段。基因转染后细胞HLE—B3经传代培养,48h后出现缓慢生长且部分细胞漂浮死亡的现象,G1期细胞明显增多。与对照细胞比较,转染后细胞的p21mRNA表达明显增多;p21蛋白表达明显增强,在经筛选的阳性克隆细胞中WB方法可检测到相对分子质量为21000的阳性蛋白带。结论 外源性p21基因可在人LEC系HLE．B3中过度表达,并对细胞周期调控产生抑制作用。基因转染抑制LEC增殖可能成为防治后发性白内障的新途径。     

Multi-layered culture of primary human conjunctival epithelial cells producing MUC5AC

The purpose of our study was to establish a system for culturing normal human conjunctival epithelial (NHCE) cells under serum-free culture conditions without compromising their ability to differentiate into a mucous epithelium. To this end, small pieces of normal conjunctiva were biopsied from patients undergoing cataract surgery. Obtained NHCE cells were cultured in bronchial epithelial growth medium (BEGM) under serum free culture conditions and passage 3 cells were air-lifted. Cultured NHCE cells displayed typical epithelial morphology. Expression of cytokeratin 19 and conjunctival epithelial specific carbohydrate residue were detected. Air-lifted NHCE cells demonstrated an increase in stratification and differentiation into goblet cells up to 3weeks under air-liquid interface (ALI) culture condition. NHCE cells expressed MUC1, MUC4, MUC16, and MUC5AC mRNA, and MUC5AC production and secretion increased in a time dependent manner after culture under ALI conditions. Exposure of cells to proinflammatory cytokines (TNF-alpha or IFN-gamma) resulted in upregulation of MUC1, MUC4, MUC16, and MUC5AC gene expression. In conclusion, differentiated NHCE cells showed features of a multi-layered conjunctival epithelium, including goblet cells, and retained functional characteristics similar to those seen in vivo. This cell culture system can better facilitate investigation of conjunctival epithelial cell biology and goblet cell differentiation.     

人原代晶体上皮细胞bFGF多肽和mRNA的表达

目的 为研究后发性白内障的发生机理,检测生长中人晶体上皮细胞（ＨＬＥＣｓ）自身是否表达碱性成纤维细胞生长因子（ｂＦＧＦ）。方法 体外培养人晶体上皮细胞,用免疫细胞化学和原位核酸分子杂交的方法,检测ＨＬＥＣｓ中ｂＦＧＦ多肽和其ｍＲＮＡ的表达。结果 用免疫细胞化学和原位核酸分子杂交方法,可检测到生长中的人晶体上皮细胞自身表达碱性成纤维细胞生长因子。结论 结合碱性成纤维细胞生长因子促进人晶体上皮细胞生长     

Role of benzalkonium chloride in DNA strand breaks in human corneal epithelial cells

Purpose

To investigate the toxic effects of benzalkonium chloride (BAC), a preservative commonly used in ophthalmic preparations, on DNA single- and double-strand breaks in immortalized human corneal epithelial cells (HCEs).

Methods

HCEs were treated with BAC in concentrations ranging from 0.00005% to 0.001% for 30?min. Cells were examined immediately after BAC exposure and after 24-h recovery. Alkaline comet assay was used to detect DNA single-strand breaks (SSBs). Immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (??H2AX) foci indicated DNA double-strand breaks (DSBs). Cell viability was measured by the MTT test.

Results

A significant increase of SSBs, detected by alkaline comet assay, was observed in a dose-dependent manner with BAC exposure in HCEs at concentrations of 0.00005% and higher. Such BAC treatment also exhibited a dose-dependent increase in DSBs, evaluated by number of ??H2AX foci. In addition, a significant change in the relative cell survival rate of HCEs was observed after exposure to 0.001% BAC for 30?min. Although the toxic effects of BAC could be partly repaired after 24?h of cell recovery, SSBs and DSBs in HCEs were still present after BAC removal.

Conclusions

The results demonstrated that exposure to BAC in HCEs, even at low concentrations, could induce DNA strand breaks, which were present after BAC removal. Cell survival analysis indicated that BAC-induced DNA damage was correlated with the cytotoxic effects.     

人眼结膜上皮干细胞的定位分析

目的探讨人眼结膜上皮干细胞的定位。方法对人眼不同部位(穹隆部、球结膜及睑结膜)结膜上皮组织用组织块培养法,种植于培养皿,观察结膜上皮细胞的生长特性。结果细胞生长速度不同:同一组织块其四周细胞生长速度不同,有的部位细胞生长快,有的部位细胞仅有移行现象;取自同一部位如穹隆部结膜的不同组织块,上皮细胞生长速度也不同,有的组织块周围细胞生长快,有的组织块周围细胞生长慢;但取自穹隆部结膜、睑结膜、角巩缘处球结膜的组织块,细胞生长形态相似。结论结膜干细胞散在分布于穹隆部和睑球结膜,但不均匀,即使在相同部位结膜干细胞分布也不平衡。