兔晶体上皮细胞培养的技术改进

对兔晶体上皮细胞培养方法作了改进，为控制后发性白内障的预防提供实验手段，取新西兰家兔晶体前囊膜作细胞培养，在原代培养中，以吸管转移囊膜，细胞融合后，用胰酶消化转代，结果：原代培养４８～７２小时后，可见晶体上皮细胞长出，以后细胞呈贴壁单层，铺砌型向外生长，传代后６～８小时细胞贴壁生长。方法经济简便，可为各种实验提供兔晶体上皮细胞。     

晶体上皮细胞单克隆抗体的动物实验研究第一部分晶体上皮细胞的培养

试图以免疫学方法制备特异性晶体上皮细胞膜单克隆抗体来抑制晶体上皮细胞的生长，以探索防止后发障的发生。共分四部分：１．晶体上皮细胞的培养；２．晶体上皮细胞膜单克隆抗体的制备；３．抗体的免疫学检测；４．所制备的单克隆抗体对动物和人的晶体上皮细胞的抑制作用。报告家兔晶体上皮细胞的原代和继代培养的方法并进行讨论。     

人晶体上皮细胞体外培养的细胞生物学特性

对体外培养的人晶体上皮细胞（LEC）进行研究，观察细胞的增生分化规律、细胞的超微结构、细胞的生化合成、物质转化及年龄等对细胞增殖的影响，为探讨老年性白内障的发病机制、对抗白内障药物的筛选、白内障术后的晶体后囊混浊形成机理以及研究细胞的衰老和细胞间亡现象等生命科学的基本问题提供理论依据．本文就此加以综述．     

晶体上皮细胞单克隆抗体的动物实验研究：第一部分晶体上皮细胞…

试图以免疫学方法制备特异性晶体上皮细胞膜单克隆抗体来抑制晶体上皮细胞的生长，以探索防止后发障的发生。共分四部分：１。晶体上皮细胞的培养；２，晶体上皮细胞膜单克隆抗体的制备；３，抗体的免疫学检测，４，所制备的单克隆抗体对动物和人的晶体上皮细胞的抑制作用。报告家兔晶体上细胞的原代和继代培养的方法并进行讨论。     

生长因子与晶体上皮细胞

白内障在致盲眼病的发生率中占首位。随着白内障囊外摘除术及人工晶体植入术的广泛开展 ,术后患者短期内视力可提高至较可观的水平。但术后 4 0 %的成年人和几乎 10 0 %的儿童发生晶体后囊混浊 (后发性白内障 ) ,导致视力再度下降。残留的晶体上皮细胞增殖是其形成原因。近年来的研究表明生长因子在促进晶体上皮细胞增殖、迁移、分化方面发挥着重要作用〔1～ 3〕。本文就生长因子与晶体上皮细胞的关系作一综述。一、生长因子概述生长因子是机体在有丝分裂原作用下 ,由体细胞产生的 ,在体内和体外对细胞的生长具有促进或抑制作用的物质。除促…     

过氧化氢对体外培养的兔晶体上皮细胞凋亡的诱导

目的研究H2O2诱导兔晶体上皮细胞凋亡.方法采用终浓度250μmol/LH2O2处理兔晶体上皮细胞,分别用TUNEL法、流式细胞仪和透射电镜检测H2O2处理后的兔晶体上皮细胞.结果用H2O2处理后的兔晶体上皮细胞,TUNEL法可见胞核呈棕褐色着染,苏木素不能复染;流式细胞仪检测到约9.6×105细胞出现亚二倍体峰,二倍体峰几近消失;透射电镜下细胞膜完整,但表面微绒毛消失,细胞质浓缩,胞浆内细胞器结构不清晰,并见大量大小不等的空泡,核质比例增大,胞核呈圆形,核膜双层结构不清,呈光滑状,核内染色质聚集成块,部分染色质边集.结论终浓度250μmol/LH2O2可以诱导兔晶体上皮细胞发生凋亡.     

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     

人晶状体上皮细胞的体外培养

目的建立一种简单可行的人晶状体上皮细胞体外培养的方法.方法利用组织块贴片法,对人晶状体的前囊膜和赤道部囊膜进行培养.对培养的细胞进行形态学观察和鉴定.结果组织块贴壁48～72h后可见人晶状体上皮细胞从组织块边缘长出,具有上皮细胞的形态特点,10～15 d后融合.在体外细胞可传五代,但第三代以后细胞表型向成纤维细胞转化.SABC法染色结果细胞胞浆内α-晶状体蛋白染色阳性.结论成功地建立起人晶状体上皮细胞体外培养模型,可用于后囊膜混浊发病机理和药物试验研究.     

细胞生长因子对人晶体上皮细胞转铁蛋白mRNA表达的影响

目的 分析转化生长因子－β２和碱性成纤维细胞生长因子对培养的人晶体上皮细胞转铁蛋白ｍＲＮＡ表达的影响,方法 人晶体前囊膜取和于３岁以下各种原因死亡２４ｈ内的２０例（４０只眼）婴和眼球。晶体上皮细胞原代培养和传代培养,选用传代汇合后２周的细胞,分别用ＴＧＦ－β１和ｂＦＧＦ作用天,用异硫氰酸胍、酚、氯仿、异戊醇等抽提细胞总ＲＮＡ,选择转铁蛋白的核酸探针进行Ｎｏｒｔｈｅｒｎ杂交,分析转铁蛋白ｍＲＮＡ的表     

晶体上皮细胞的功能和形态学研究

晶体上皮细胞层是晶体组织内代谢最活跃的部位。晶体上皮细胞间的紧密连接和正常形态是保证其功能正常、维持晶体组织均匀透明的基础。晶体上皮细胞的功能损害和形态改变，必然导致晶体的混浊。后发性白内障主要是残留的晶体上皮细胞在囊膜上异常增殖、移行的结果。本对晶体上皮细胞的功能和形态以及白内障，特别是后发性白内障时晶体上皮细胞的功能和形态学变化进行综述。     

盖玻片辅助人晶状体上皮细胞原代培养法

目的：建立人晶状体上皮细胞原代培养的简便方法并比较不同来源人品状体上皮细胞的生物学特性。方法：取胎龄20周合法引产胚胎眼晶状体囊膜、中山眼科中心眼库眼晶状体囊膜和白内障患者术中撕取的前囊膜，分别在培养皿中铺平，加10乩10％DMEM培养液润湿后加盖盖玻片防止卷曲并促进粘贴．添加培养液浸没盖玻片，37℃培养。同时取相同来源的囊膜按照组织块法培养。观察细胞增殖情况并比较原代人晶状体上皮细胞与人晶状体上皮细胞系SRA01／0413晶体蛋白的表达差异。结果：在盖玻片辅助下，胚胎眼晶状体囊膜第2天即可见明显的增殖细胞由囊膜缘长出，眼库眼囊膜和白内障患者术中撕取的囊膜在3～4d的潜伏期后亦可见增殖细胞长出；组织块法培养出现部分组织块漂浮，且胚胎眼囊膜潜伏期延长至3-4d，眼库眼囊膜和白内障患者晶状体囊膜潜伏期延长至4-5d。结论：盖玻片辅助的改良组织块培养法能尽快获得体外培养的原代晶状体上皮细胞，且操作简便，值得推广应用于品状体病的研究。     

The Biological Study of the Cultured Human Lens Epithelial Cells in Vitro

The human lens epithelial cells (HLE) cultured in vitro was established in normal and cataractous lenses. The biological feature, histological characteristics and the ultrastructure of the cultured HLE cells were investigated. The results reveal that the proliferative capacity of the culutured HLE cells is reversely proportional to the donour age; the cultured HLE cells has the limited proliferative capacity in vitro. The relieve of the contact inhibition is the effective trigger of the HLE cell proliferation. This research work is possible to make a basis for the further study of the regeneration of the lens and the roles of the lens epithelial cells in the development of the cataract. Eye Science 1994; 10:27-31.     

Effects of Calcium on Human Lens Epithelial Cells In Vitro

Purpose To analyze the effect of calcium on human lens epithelial cells (LECs) in vitro.Methods Human LECs were obtained from the anterior lens capsule during a cataract operation, and were cultured in minimum essential medium (MEM) containing 12% fetal bovine serum for a week at 37°C, 5% CO₂. The LECs were then isolated with trypsin and placed in culture dishes. To analyze the effects of calcium, the LECs were incubated in MEM with calcium concentrations of 0, 2, 10, or 20mM. After H&E staining for 72h, the LECs were analyzed with the Scion imaging program.Results The LECs proliferated rapidly and their shapes were uniform in 2-mM-calcium MEM. The LECs proliferated more slowly and had irregular shapes in MEM with lower and higher concentrations of calcium.Conclusions Calcium affects both the proliferation and the shape of human LECs in culture. Abnormal (hyper- or hypo-) calcium concentrations in the lens and aqueous humor may change the homeostasis of LECs, resulting in cataracts and secondary posterior capsular opacification. Jpn J Ophthalmol 2004;48:97–100 © Japanese Ophthalmological Society 2004     

高三尖杉酯碱对兔眼晶体上皮细胞的作用

目的:研究高三尖杉酯碱(Hh)对兔眼晶体上皮细胞(LEC)的作用,以探讨白内障防治的可能性。方法:在9只新西兰大白兔的右眼(用药眼)作晶体超声乳化术时,用含有Hh(20μg/ml)的平衡盐溶液作灌注液。除了术前术后眼部检查以外,并于术后30分钟、3天和60天取出眼球作LEC的光学和电子显微镜检查,同一兔子的左眼(对照眼)作晶体超声乳化术时使用平衡盐溶液作灌注液。结果:手术后3天用药眼晶体上皮细胞的增殖明显轻于对照眼。虽然在手术后60天双眼均出现后发白内障-Soemmering环,但用药眼晶体上皮细胞可见细胞浆有空泡形成等超微结构改变。结论:提出如果在晶体超声乳化术时增加Hh在灌注液的含量或延长其在房水的停留时间,可能增加对晶体上皮细胞的抑制作用。此初步研究在探索后发性白内障方面具有一定的意义。眼科学报 1995;11:192—196。     

添加晶体皮质提高晶体上皮细胞原代培养成功率

目的介绍一种提高晶体上皮细胞原代培养成功率的方法。方法基本步骤为将一晶体前囊膜剪碎后加纯胎牛血清０．５ｍｌ和冰冻过的新生牛晶体皮质０．５～１ｍｌ，密封培养２４～４８ｈ后添加约３ｍｌ含１０％胎牛血清的ＤＭＥＭ培养液继续培养。结果用该方法原代培养新生牛、幼年兔和人胚胎及角膜移植后的中青年供体标本，成功率为１００％，老年白内障手术取出标本成功率为８７％。结论添加晶体皮质可提高晶体上皮细胞原代培养成功率。     

人角膜上皮细胞的体外培养方法探讨

目的:探寻角膜上皮移植细胞的体外培养方法。方法:用改良的细胞悬液培养法与组织块培养法,描绘两种方法获取细胞的生长曲线,并计算细胞倍增时间;用~3H-胸腺嘧啶核苷(~3H-TdR)掺入与液体闪烁技术观察细胞DNA合成能力。结果:改良的细胞悬液法与组织块培养法培养的细胞平均倍增时间分别为54.15± 4.28小时和67.68±1.96小时(P<0.01);应用前者培养的细胞DNA合成也明显活跃(P<0.01)。结论:从角膜缘取材,应用改良的细胞悬液培养法收集的角膜缘上皮细胞含有角膜上皮干细胞,具有更强的分裂增殖能力,更适于体外培养的角膜上皮细胞移植之用。也证实了角膜上皮干细胞存在于角膜缘基底层的观点。眼科学报 1997;13:67～69。     

Lens Epithelial Cell Proliferation and Cell Density in Human Age—relat

Purpose: To discuss the potential effect of the lens epithelial cell proliferation inage-related cataract.Methods: In vitro cell proliferation was assayed by MTT method to evaluate the lensepithelial cell density, index, and proliferation capacity in normal lens and all kinds ofage-related cataract. Capsulotomy specimens from all kinds of patients who underwentcataract phacoemulsification extraction surgery were compared with the lens epithelialspecimens from non-cataract lenses of Eye Bank eyes.Results:Lens epithelial cell density of central anterior capsule(LECD) in female normallens was higher than that in male, LECD in nuclear cataract (> NⅢ) was higher thanthat in normal lens, but in the mature cortical cataract, LECD was lower. Mitotic indexof three kinds of age-related cataracts in vivo had no statistical difference, neither didcell proliferation capacity of cultivated cells in vitro.Conclusion: The individual difference of lens epithelial cell density and proliferationcapacity in vivo may be an     

Cytotoxicity of Contact Lens Care Solution to Human Corneal Epithelium in Vitro

Purpose: To investigate the cytotoxicity of soft contact lens multi-purpose care solutions which are now in common use in China.Methods: The cell culture method was used. Cytotoxicity was indicated by significant increases in the number of dead cells relative to controls.Results: Cells were exposed to soft contact lens care solutions for 15 min. They were irregular in shape and variable in size. The intercellular space increased and variable in size.The in-tercelluar space increased and the cells became scrunken. With the time of exposure elongated , damage of cells became more severe.Conclusions: Four kinds of soft contact lens multi-purpose care solutions may have harmful effects on the culture of human corneal epithelial cells. Soaked lenses should be rinsed with saline before being placed in the eyes in order to reduce the potential toxicity of contact lens care solutions. Eye science 1998; 14 :45 - 47.     

Proteomic Analysis of Human Lens Epithelial Cells Exposed to Microwaves

Purpose

To study proteomic changes in human lens epithelial cells (HLECs) exposed to 1800-MHz Global System for Mobile Communication (GSM)-like microwaves.

Methods

In three separate experiments, HLECs were exposed and sham-exposed (six dishes each) to 1800-MHz GSM-like radiation for 2?h. The specific absorption rates were 1.0, 2.0, or 3.5?W/kg. Immediately after radiation, the proteome was extracted from the HLECs. Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis(2-DE; silver staining) and PDQuest 2-DE analysis software were used to separate and analyze the proteome of exposed and sham-exposed HLECs. Four differentially expressed protein spots were selected and identified by using electrospray ionization tandem mass spectrometry (ESI-MS-MS).

Results

When the protein profiles of exposed cells were compared with those of sham-exposed cells, four proteins were detected as upregulated. After analysis by ESI-MS-MS and through a database search, heat-shock protein (HSP) 70 and heterogeneous nuclear ribonucleoprotein K (hnRNP K) were determined to be upregulated in the exposed cells.

Conclusions

Two-dimensional polyacrylamide gel electrophoresis combined with mass spectrometry may be a powerful tool for screening potential electromagnetic-reaction protein markers. HSP70 and hnRNP K are involved in the stress reaction of HLECs exposed to microwaves. These cell responses are nonthermal effects of the electromagnetic field.?Jpn J Ophthalmol 2007;51:412–416 © Japanese Ophthalmological Society 2007

     

体外培养视网膜色素上皮细胞对成纤维细胞生长的促进作用

观察体外培养的视网膜色素上皮细胞条件培养液对成纤维双细胞生长的影响。利用＾３Ｈ胸嘧啶核苷掺入法和 细胞计数法首次显示了视网膜色素上此细胞条件培养液对成纤维细胞生长有促进作用。