兔结膜成纤维细胞培养的体会

兔结膜成纤维细胞培养的体会河北省人民医院张坤丽，张淑惠，杨树立，张渤海近年来，抗眼部增生性疾病的研究已进入抑制细胞增殖的药物研究领域，其中，体外抗成纤维细胞药物测试则成为一种重要的研究工具［１，２］。本文就我们培养兔结膜成纤维细胞的体会介绍如下。材料...     

兔结膜成纤维细胞的体外培养

目的：探索兔眼结膜成纤维细胞体外培养的方法。方法：分别采用组织块培养法、机械分离法、消化培养法、细胞悬液法和混合消化组织块培养法培养兔结膜成纤维细胞,观察细胞形态和生长特性。结果：单纯机械分离和消化培养法都可以得到单层贴壁细胞,细胞消化后再培养可缩短生长周期。结论：混合酶充分消化的组织块连同细胞悬液一同培养的方法,步骤简便且周期短。通过差别消化法和多次传代可以得到较纯的成纤维细胞。     

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     

依地酸二钠在兔结膜成纤维细胞体外培养中的应用

目的：研究EDTA溶液在体外培养兔结膜成纤维细胞的应用。方法：将无菌条件下取得的兔结膜用2.5g/L的胰蛋白酶消化后，置于50mL/L CO2、37℃培养箱中，在细胞贴壁长满后以0.4g/L EDTA溶液消化，传代、接种，共18瓶。兔的另1眼结膜用2.5g/L的胰蛋白酶作传代消化液，同样条件培养18瓶。接种48h后观察2组结果。结果：EDTA组兔结膜成纤维细胞生长良好，胰蛋白酶组18瓶中有6瓶生长不良。结论：0.4g/L EDTA溶液用于兔结膜成纤维细胞培养传代时的消化，可减少消化时对细胞的损伤。     

依地酸二钠在兔结膜成纤维细胞体外培养中的应用

目的研究EDTA溶液在体外培养兔结膜成纤维细胞的应用。方法将无菌条件下取得的兔结膜用2.5g/L的胰蛋白酶消化后,置于50mL/CO2、37℃培养箱中,在细胞贴壁长满后以0.4g/L EDTA溶液消化,传代、接种,共18瓶。兔的另1眼结膜用2.5g/L的胰蛋白酶作传代消化液,同样条件培养18瓶。接种48h后观察2组结果。结果EDTA组兔结膜成纤维细胞生长良好,胰蛋白酶组18瓶中有6瓶生长不良。结论0.4g/L EDTA溶液用于兔结膜成纤维细胞培养传代时的消化,可减少消化时对细胞的损伤。     

汉防己甲素对体外培养的兔结膜成纤维细胞抑制作用的实验研究

目的：观察汉防己甲素（Tet)对体外培养的兔结膜成纤维细胞（RCF）生长的抑制作用，探讨其作用的可能机制，为防治与成纤维细胞增殖相关的多种眼病提供一种有效的药物。方法：应用MTT法测定不同浓度的Tet对RCF的抑制作用；用细胞计数法观察不同浓度的Tet在不同时间下对RCF的生长抑制作用；用流式细胞仪检测Tet对RCF细胞周期的影响。结果：给药48小时后，终浓度为0．16、0．8、4、20、100μg/ml的Tet对RCF的生长均明显抑制作用；且在上述不同浓度的Tet作用下，于1、3、5、7天计数细胞，发现Tet对RCF的抑制具有时间、剂量关系。流式细胞仪显示，RCF经2μ/mlTet处理48h后，G1期细胞增加了13．3％，S期细胞下降了9．55％，G2期细胞下降了4．23％。结论：汉防己甲素可抑制RCF生长，有时间剂量效应。Tet影响RCF细胞周期各时相群体，抑制G1期向S期的过渡，进而阻止细胞向G2期的过渡，从而抑制细胞增殖。     

结膜松弛症球结膜成纤维细胞培养的研究

目的：为了筛选适合结膜松弛症结膜成纤维细胞培养的方法,对不同培养基培育及杞精明目汤药物血清作用前后的结膜松弛症和正常球结膜成纤维细胞进行观察。 方法：收集结膜松弛症松弛结膜组织16例、翼状胬肉组织11例和正常球结膜组织5例,观察成纤维细胞的生长状态,筛选最适合结膜松弛症成纤维细胞生长的培养基。 结果：采用DMEM/F12培养基可使结膜松弛症成纤维细胞少量从组织块内溢出,但不能传代。采用含100mL/L胎牛血清、1μL/mL成纤维细胞生长添加物（FGS）的DMEM-H培养基可使结膜松弛症成纤维细胞顺利生长,且能够传代。 结论：最适宜培养结膜松弛症成纤维细胞的培养基是含100mL/L胎牛血清、1μL/mL FGS、100U/mL青霉素、100μg/mL链霉素的DMEM-H培养基。     

反义c—myc寡核苷酸对培养的人结膜成纤维细胞增生的抑制作用的观察

目的：研究反义寡核苷酸能否对闭原癌基因c-myc的表达并抑制培养的人结膜成纤维细胞的增生,为青光眼滤过术后滤过泡的瘢退化探索新的治疗途径。方法：取人结膜下筋膜进行成纤维细胞的培养,脂质体Lipofectamine^TM 2000的介导下分别将正义c-myc,反义c-myc转入细胞中,MTT法检测其对细胞增生活性的影响,RT－PCR扩增检测其对细胞mRNA表达水平的影响。结果：反义c-myc能显抑制培养的人结膜成纤维细胞的增生,并抑制其c-myc mRNA的表达,而正义c-myc无此作用,反义c-myc的最适浓度为2uM。结论：反义c-myc通过阻断人结膜成纤维细胞中原癌基因c-myc表达来抑制细胞的增生。     

汉防己甲素体外对正常结膜与翼状胬肉成纤维细胞抑制作用的比较

目的:观察汉防己甲素(tetrandrine,Tet)对正常结膜和翼状胬肉成纤维细胞抑制作用的异同。方法:在相同的培养条件下,传第3代的正常结膜与翼状胬肉成纤维细胞皆加入不同浓度的Tet,分别于加药后1和3d行MTT检测细胞的存活率。结果:用药后1d,4×10-5和2×10-5mol/LTet对正常结膜与翼状胬肉成纤维细胞存活率无显著性差异(P>0.05),其余各浓度的Tet对翼状胬肉成纤维细胞的抑制作用大于正常结膜成纤维细胞,正常结膜成纤维细胞的存活率大于翼状胬肉成纤维细胞(P<0.01)。用药后3d,4×10-5mol/LTet组翼状胬肉成纤维细胞的存活率大于正常结膜成纤维细胞(P<0.01);其余各浓度的Tet对两种成纤维细胞存活率无显著性差异(P>0.05)。结论:用药后3d,2×10-5mol/L及半数抑制量(10-5mol/L)以下浓度的Tet对翼状胬肉成纤维细胞起抑制作用,而对正常结膜成纤维细胞的影响较小。     

汉防己甲素及地塞米松对兔结膜成纤维细胞增殖的抑制作用

目的　观察汉防己甲素 (tetrandrine,Tet)对体外培养的兔结膜成纤维细胞 (rab-bit conjunctival fibroblasts,RCF)增殖的抑制作用 ,探讨其与地塞米松 (dexamethasone,Dex)联合应用的价值 ,并对二者的抑制作用进行比较。方法　应用 MTT法 ,测定不同浓度的 Tet、Dex及 Tet联合 Dex对 RCF增殖的抑制作用。结果　浓度为 1 0 0、80、2 0、5、1 .2 5、0 .3 1 2 5mg· L-1的 Tet对 RCF的增殖均有抑制作用 ,其抑制率分别为 86.0 0 %、78.1 5%、62 .89%、3 0 .1 9%、1 8.0 4%、9.2 1 %,与对照组相比有显著性差异。半数抑制量(ID50 ) 2 0 0 mg·L-1的 Dex在联合 5mg· L-1的 Tet后 ,对细胞生长的抑制率为 49.2 5%.结论　 Tet对 RCF的增殖有抑制作用 ,且与药物浓度成正相关。 Dex联合 Tet有协同抑制作用。 Tet可能成为一种新型的抗眼部增殖性疾病的药物     

SQUAMOUS CELL CARCINOMA OF THE CONJUNCTIVA: CASE PRESENTATION AND TREATMENT

A case of the rare condition of invasive squamous cell carcinoma of the conjunctiva is presented The plan for surgical excision of the area involved, determined by resultsof frozen section biopsies , is outlined The methods of treatment of other authors are briefly reviewed, including the method (from Fraunfelder) of combined excision and cryotherapy .     

AN ULTRASTRUCTURAL STUDY OF THE ROLE OF LEUCOCYTES AND FIBROBLASTS IN THE BREAKDOWN OF BRUCH'S MEMBRANE

Ultrastructural examination was performed on 30 eyes selected on the basis of a light microscopic classification as representing all stages of macular degeneration. The eyes belonged to 29 patients aged 40 to 91 years at death and all patients with the exception of the 40-year-old had been examined during life. The results indicate that monocytes, lymphocytes, fibroblasts and mast cells may play a role in the formation of breaks in Bruch's membrane at early and late stages of macular degeneration. The implications of these observations are discussed in the context of their possible role in choroidal neovascularization.     

VASOACTIVE INTESTINAL POLYPEPTIDE IMMUNOREACTIVE NERVE FIBRES IN THE HUMAN EYE

Immunohistochemistry applied to whole-mount preparations was used to investigate the presence and distribution of vasoactive intestinal polypeptide (VIP) immunoreactive nerves in the non-retinal part of the human eye. The choroid has a dense perivascular supply of VIP immunoreactive nerve fibres, and some free nerve endings within the stroma. These nerves enter the choroid in ciliary nerves and also as perivascular networks around the ciliary arteries. Occasional choroidal VIP immunoreactive nerve cell bodies are seen. The ciliary body stroma, close to the iris root has a dense circumferential plexus of VIP immunoreactive nerve fibres that occur both singly and in bundles. The iris root has a circumferential arrangement of bundles from which VIP immunoreactive nerve fibres travel radially in the stroma. They supply the pupillary region with numerous free nerve endings: the sphincter pupillae is not supplied by these nerves. The cornea is devoid of VIP immunoreactive nerves. These findings, together with existing knowledge of the physiological actions of VIP, indicate that VIP immunoreactive nerves are likely to be involved in the functioning of several ocular tissues.     

人三叉神经节细胞体外培养及对HSV—1感染敏感性的实验研究

首次报告了人三叉神经节Trigeminal Ganglian(TG)三种原代培养细胞在体外对单纯疱疹病毒1型(HSV-1)Mckrae株感染后的不同时相的敏感性实验研究。结果表明人TG中的神经节细胞对HSV-1感染最敏感,HSV—1抗原可以在其胞浆及胞核中迅速出现。成纤维细胞和胶质细胞也有部分感染现象。这种结果提示TG中的神经节细胞很可能是HSV-1主要的潜伏感染细胞。     

神经营养因子与生长因子在体外对人视网膜神经节细胞之作用

目的 研究神经营养因子(neurotrophic factor)与生长因子(growth factor)在体外对人视网膜神经节细胞(retinal ganglion cell,RGC)生存的作用。 方法 从捐献的尸体眼球分离并培养RGC。RGC之鉴定系根据形态学与免疫细胞化学研究。将各种神经营养因子与生长因子分别加入培养液。对RGC计数,并与未加任何因子的对照组相比较。 结果 未加任何因子的培养中不见或仅有极少量的RGC。加有神经营养蛋白-3(neurotrophin-3,NT-3)、神经生长因子(nerve growth factor,NGF)、表皮生长因子(epidermal growth factor,EGF)与血小板源性生长因子(plate derived growth factor, PDGF)的培养亦同。加有下列因子的培养中有较多的RGC出现,计数为(细胞数/每10个显微镜野)：脑源性神经营养因子(brain derived neurotrophic factor,BDNF)：4.08;睫状神经营养因子(ciliary neurotrophic factor,CNTF)：1.23;神经营养蛋白-4/5(neurotrophin-4/5,NT-4/5)：2.63;碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)：2.65。 结论 BDNF、NT-4/5、bFGF与CNTF在体外能改善人RGC之生存,而NGF、NT-3、EGF与PDＧF则不能。 (中华眼底病杂志, 1999, 15: 149-152)     

THE USE OF SPECULAR MICROSCOPY TO INVESTIGATE UNUSUAL FINDINGS IN THE CORNEAL ENDOTHELIUM AND ITS ADJACENT STRUCTURES

The use of specular microscopy is not confined to the corneal endothelial mosaic, but may be used at any level through the corneal stroma to the posterior endothelial surface. In the stroma the not very dense, irregularly rounded deposits of corneal dystrophies may be differentiated from the dense, geometric shapes of crystalline deposits. At the level of Descemet's membrane the fine parallel lines of tears in Descemet's are distinct from the multiple fine vertical deep stromal lines seen in advanced keratoconus. At the level of the endothelial mosaic the cells may be counted and their morphology examined, while blebs may also be seen in a wide variety of pathological conditions including superficial keratopathies, contact lens wearers, deep keratopathies, anterior uveitis and contusion injury. These blebs vary in size in different conditions and are often transient. They must be distinguished from guttae which are permanent and although an occasional finding in normal corneas are a constant feature of cornea gutatta and Fuchs' dystrophy. On the posterior endothelial surface the relief mode enables the examination of numerous deposits including red blood cells, white blood cells, keratitic precipitates, pigment granules and pseudoexfoliative KP. The morphology of these various findings is discussed.     

RETINAL DEVELOPMENT IN HUMANS: THE ROLES OF DIFFERENTIAL GROWTH RATES, CELL MIGRATION AND NATURALLY OCCURRING CELL DEATH

The distribution of ganglion cells throughout the retinal ganglion cell layer is non-uniform in adult mammals. This paper reviews some of our data describing the development of retinal ganglion cell topography in the human fetus. Results indicate that early in the fetal period the distribution of ce/fe in the ganglion cell layer is almost uniform, but by the end of gestation there is a gradient in cell density of about 10:1 (centrahperipheral). Peripheral retina grows more rapidly than the central retina prior to about 23 weeks gestation, but this differential growth rate apparently has little effect on the development of a centre-peripheral density gradient. The gradient appears between about 18 and 30 weeks gestation, and during this period there appears to be a greater rate of cell death in the ganglion cell layer of the peripheral retina. Cell density at the developing fovea is less than the perifoveal cell density at all ages, suggesting that ganglion cells migrate from fovea/ into perifoveal regions throughout the fetal period.     

THE IDENTIFICATION OF DIFFERENT VASCULAR CELLS ON THE CORNEAL ENDOTHELIUM BY SPECULAR MICROSCOPY I. RED BLOOD CELLS AND CORNEAL ENDOTHELIAL CHANGES IN HYPHAEMA SECONDARY TO CONTUSION INJURY

In order to differentiate clearly red blood cells and their relationship to the corneal endothelium, specular microscopy was performed in patients with hyphaema due to contusion injury. This enabled the clear identification of red blood cells as rounded, umbilicated or flattened spheroidal structures depending on whether they were seen side on or end on. They were seen best in the relief mode lying on the posterior endothelial surface and not infiltrating the corneal endothelium. It also disclosed the presence of subendohelial blebs one to three cells in diameter seen at the level of the corneal mosaic and not in the relief mode. These are probably due to mild oedema following the contusion injury and were distinct from the red blood cells. The corneal endothelial cell count and morphology were similar to that in the other eye when the changes resolved a week or so after the injury.