猴,兔角膜细胞成分原代培养的研究

目的 建立从同一角膜上上分离培养角膜上皮,基质和内皮细胞的方法。方法 采用消化法培养猴角膜内皮细胞,上皮细胞和兔角膜上皮细胞,组织块培养法培养角膜基质细胞和兔角膜内皮细胞,结果 猴,兔角膜内皮细胞培养１周后,均能形成细胞单层,猴角膜上皮细胞培养３－４天生长旺盛,但难以形成细胞单层,兔上皮细胞生长旺盛,１周后已达融合状态,细胞呈膜状伸出板层伪足,猴,兔基质细胞易培养,１周后融合成单层细胞,结论 从同     

同一猴、兔眼角膜细胞成分原代培养的实验研究

目的建立从同一角膜片上分离培养角膜上皮、基质和内皮细胞的方法,为研究角膜细胞间的相互作用机制奠定基础。方法采用消化法分离培养猴眼角膜内皮细胞、上皮细胞和兔眼角膜上皮细胞．组织块培养法培养基质成纤维细胞和兔角膜内皮细胞。细胞接种于12孔培养板,并于培养不同时间行Wright染色检查细胞生长情况。结果采用消化法和组织块培养法能成功地培养猴、兔角膜上皮、基质和内皮细胞。猴、兔角膜内皮细胞培养1wk后,均能形成内皮细胞单层,细胞类似天然的六角形态．且以猴内皮细胞明显;猴角膜上皮细胞培养3～4d生长旺盛、但难以形成细胞单层,兔上皮细胞生长旺盛,1wk后已达融合状态,细胞呈膜状伸出板层伪足;猴、兔基质成纤维细胞易培养,1wk后已融合成单层细胞,细胞排列整齐,类似纤维走行样外观。结论从同一角膜材料中能分别培养出角膜3种细胞成份,消化法和组织块培养法相结合既能节省材料,又简单易行。     

二肽基肽酶II在正常大鼠眼球各组织中的免疫表达

目的:探讨二肽基肽酶II(dipeptidyl peptidaseII,DPPII)在正常大鼠眼球各组织中的免疫表达。方法:应用抗生物素蛋白-生物素-过氧化物酶复合体法,检测DPPII在正常大鼠眼球各组织中的免疫表达。结果:发现DPPII在角膜上皮细胞、角膜基质层、角膜内皮细胞、睫状体无色素上皮细胞、虹膜色素上皮细胞、晶状体上皮细胞、晶状体纤维、视网膜神经纤维层、神经节细胞、视网膜色素上皮细胞中免疫染色均呈阳性。结论:DPPII存在于角膜上皮细胞、角膜基质层、角膜内皮细胞、睫状体无色素上皮细胞、虹膜色素上皮细胞、晶状体上皮细胞、晶状体纤维、视网膜神经纤维层、神经节细胞、视网膜色素上皮细胞。     

二肽基肽酶II在正常大鼠眼球各组织中的免疫表达

目的:探讨二肽基肽酶II(dipeptidyl peptidaseII,DPPII)在正常大鼠眼球各组织中的免疫表达。方法:应用抗生物素蛋白-生物素-过氧化物酶复合体法,检测DPPII在正常大鼠眼球各组织中的免疫表达。结果:发现DPPII在角膜上皮细胞、角膜基质层、角膜内皮细胞、睫状体无色素上皮细胞、虹膜色素上皮细胞、晶状体上皮细胞、晶状体纤维、视网膜神经纤维层、神经节细胞、视网膜色素上皮细胞中免疫染色均呈阳性。结论:DPPII存在于角膜上皮细胞、角膜基质层、角膜内皮细胞、睫状体无色素上皮细胞、虹膜色素上皮细胞、晶状体上皮细胞、晶状体纤维、视网膜神经纤维层、神经节细胞、视网膜色素上皮细胞。     

二肽基肽酶Ⅲ在正常大鼠眼球中的免疫表达

目的 探讨二肽基肽酶Ⅲ(dipeptidyl peptidase Ⅲ,DPP Ⅲ)在正常大鼠眼球各组织中的免疫表达.方法 选取正常的两性Wistar大鼠,应用抗生物素蛋白-生物素-过氧化物酶复合体法检测DPP Ⅲ在正常大鼠眼球各组织中的免疫表达.结果 DPP Ⅲ在角膜上皮细胞、角膜内皮细胞、睫状体无色素上皮细胞、虹膜色素上皮细胞、晶状体上皮细胞、晶状体纤维细胞、视网膜神经纤维层、视网膜神经节细胞、视网膜外丛状层、视网膜色素上皮细胞中免疫组织化学染色均呈阳性.结论 DPP Ⅲ主要存在于Wistar大鼠眼球的角膜上皮细胞、角膜内皮细胞、睫状体无色素上皮细胞、虹膜色素上皮细胞、晶状体上皮细胞、晶状体纤维细胞、视网膜神经纤维层、神经节细胞、视网膜外丛状层、视网膜色素上皮细胞.     

体外培养中牛角膜内皮细胞和虹膜色素上皮细胞的

目的明确FasL在体外培养的角膜内皮细胞和虹膜色素上皮细胞（IPE）中FasL的表达情况及其意义。材料和方法采用酶辅助的显微分离法分离新生牛眼IPE细胞,酶消化法分离角膜内皮细胞,进行体外细胞培养。抗FasL抗体、S-P试剂盒（DAB显色）对传一代IPE细胞和角膜内皮细胞进行免疫组化染色。结果角膜内皮细胞FasL染色呈阳性;IPE细胞及对照组均为阴性。结论在前房免疫赦免的形成中,角膜内皮细胞主要通过表达和分泌FasL发挥作用,而IPE细胞则主要通过表达和分泌有免疫抑制作用的细胞因子以及通过一种非Fas-FasL诱导细胞凋亡的通路来阻止T细胞活化、增殖。     

羊膜载体培养标记兔角膜内皮细胞移植的研究

目的探讨以羊膜基底膜为载体培养兔角膜内皮细胞移植的可能性,为培养内皮细胞移植治疗角膜内皮失代偿疾病提供依据与方法。方法体外培养扩增兔角膜内皮细胞,采用亲脂性碳青染料(CM-DiI)对细胞进行标记,种植于去除上皮细胞的羊膜基底膜上,体外培养形成单层角膜内皮层,并进行形态学、组织学、超微结构及细胞标记情况的观察;将羊膜为载体培养的内皮层对切除后板层的兔角膜进行移植,同时以无培养内皮细胞的羊膜移植和单纯角膜后板层切除为对照,术后观察角膜透明度与厚度,对其进行组织学与细胞标记情况的检测。结果 5～7d 角膜内皮细胞即在羊膜基底膜上融合成单层,细胞为扁平多角形,排列紧密,密度可达(3202.84±347.77)个/mm~2,荧光显微镜下可见内皮细胞被 CM-DiI 标记后显现红色荧光;培养内皮层移植后角膜维持相对的透明与薄度,而无内皮细胞羊膜移植和单纯后板层切除两组对照角膜严重水肿、混浊,厚度明显超过实验组角膜;培养内皮移植后角膜形成新的内皮层,通过标记的细胞发现移植后细胞仍为培养移植的内皮细胞而非周边细胞的移行。结论羊膜基底膜是角膜内皮细胞生长和移植的良好载体,体外培养角膜内皮细胞移植有望代替供体角膜移植,具有广阔的应用前景。(中华眼科杂志,2006,42:925-929)     

体外培养中牛角膜内皮细胞和虹膜色素上皮细胞的FasL表达

目的：明确FasL在体外培养的角膜内皮细胞和虹膜色素上皮细胞（IPE）中FasL的表达情况及其意义,材料和方法：采用酶辅助的显微分离法分离新生牛眼IPE细胞,酶消化法分离角膜内皮细胞,进行体外细胞培养,抗FasL染色呈阳性;IPE细胞及对照组均为阴性。结论：在前房免疫赦免的形成中,角膜内皮细胞主要通过表达和分泌FasL发挥作用,而PE细胞则主要通过表达和分泌有免疫抑制作用的细胞因子以及通过一种非FasL－FasL诱导细胞凋亡的通路来阻止T细胞活化、增殖。     

兔角膜内皮、上皮及基质细胞体外培养扩增的研究

目的 建立角膜上皮、基质及内皮细胞体外培养扩增的简单稳定的方法，为组织工程化角膜的构建提供种子细胞。方法 内皮细胞与后弹力层在培养基中孵育后消化法获原代细胞，胰酶消化去除表层上皮后取角膜缘，组织块法培养角膜缘上皮细胞，基质细胞应用胶原酶消化法获原代培养，各细胞融合后胰酶消化依次传代培养。结果 原代内皮细胞4—5d融合成单层细胞，可连续传6—7代。上皮细胞1周左右生长融合，连续传3—4代后细胞形态改变。基质细胞接种6—7d后近融合，传代后增殖明显，可连续传10代。结论依据角膜组织特征选择合适的方法体外分离、培养角膜3种细胞成分，可获连续传代扩增的角膜细胞。     

应用三维培养技术体外重建角膜组织的初步研究

目的 探讨三维培养技术体外重建角膜组织的理论基础及临床应用价值。方法 原代培养人角膜上皮、基质及内皮细胞、通过胶原凝胶三维培养系统,体外重建角膜结构,应用裂隙灯检测人工角膜的透明度,采用常规病理组织学及透射电镜观察细胞在三维培养系统中生长的特性。结果 重建的角膜组织为良好的三层结构;角膜上皮细胞形成复层结构;内皮细胞形成单层结构,且排列规则;角膜基质细胞呈散在分布。电镜观察、细胞在Ⅰ型胶原中存活并合成胶原纤维。结论 利用组织工程技术,能够体外重建结构较规则,透明度较高的角膜组织。     

Adult retinal neuronal cell culture

Despite a relatively long history, general knowledge is not widespread that adult neurons can be maintained in cell culture for fairly extended periods of time. Within the central nervous system, this capacity seems to be particularly well developed in the retina, although it is still not clear whether this property is due to physical reasons (spatial configuration, simple connections) or to more fundamental differences (molecular composition, physiological function). Irrespective of the reasons, in vitro model systems are useful for investigating physiological and pathological processes occurring in mature retina. The authors argue that the numerous molecular changes undergone during maturation (modifications in ion channels and receptors, apoptotic pathways and growth factor effects) should be taken into account when using in vitro approaches to study processes involved in photoreceptor and ganglion cell degeneration, and hence that more classical methods relying on embryonic or newborn tissue should be interpreted with caution. A number of examples are given where the use of adult retinal neuronal culture may be especially informative: neurite regeneration, neuroprotection assays and pathogenic mechanisms; and areas of further research that should be explored: cell transplantation.     

Corneal deturgescence during organ culture

A simple method is described to monitor rabbit and human corneal deturgescence while the tissue is suspended in tissue culture media. The composition of the media is such that the cornea slowly thickens at 4 degrees but exhibits the classic "temperature reversal" thinning phenomenon when incubated at 23 degrees-37 degrees. Each cornea is cultured in a closed eye bank corneal viewing chamber during swelling and deswelling phases of an experiment, minimizing direct handling of the tissue. Optimal conditions for observing corneal deturgescence are described. This protocol has several advantages over corneal perfusion techniques and could be further developed for routine use in eye banks as a functional test of donor suitability for keratoplasty.     

Premacular membranes in tissue culture

Graefe's Archive for Clinical and Experimental Ophthalmology - To investigate integrity and characteristics of human premacular membranes (PMM) with and without standard tissue culturing using...     

Efficiency of blood culture bottles for the fungal sterility testing of corneal organ culture media

BACKGROUND/AIM: The consequences of fungal contamination of an organ cultured cornea, though exceptional, are often disastrous for the recipient. Consequently, eye banks often quarantine corneas for 10 days or more before passing them for grafting. This period, though detrimental to the endothelial cell density of the delivered cornea, is necessary to detect contamination using conventional microbiological methods. The authors previously validated the use of a pair of aerobic and anaerobic blood bottles for sensitive and rapid detection of bacteria. To allow a short quarantine period, it remained only to optimise detection of fungi. The authors aimed to compare sensitivity and rapidity of fungal contamination detection by three methods: blood bottles, Sabouraud, and daily visual inspection of the organ culture medium. METHODS: Four inocula (10(6), 10(4), 10(2), 10 colony forming unit (CFU) per ml) of 11 fungi (Candida albicans, C tropicalis, C glabrata, Saccharomyces cerevisiae, Rhodotorula rubra, Cryptococcus neoformans, Fusarium oxysporum, Aspergillus niger, A fumigatus, A flavus, Acremonium falciforme) were inoculated in a commercial organ culture medium containing a coloured pH indicator (CorneaMax, Eurobio, Les Ulis, France). The real live fungal inoculum was verified immediately after inoculation. After 48 hours at 31 degrees C, samples of the contaminated media were inoculated in three blood bottles: Bactec Aerobic/F, Bactec Mycosis IC/F, and Bactec Myco/F Lytic (Becton Dickinson, Le Pont de Claix, France), then placed in a Bactec 9240 rocking automat, and in four Sabouraud media (solid and liquid, 28 degrees C and 37 degrees C) with daily observation. Contaminated organ culture media were also checked daily for any change in turbidity and/or colour. Experiments were performed in triplicate. RESULTS: Mycosis IC/F and Myco/F Lytic bottles were neither faster nor more sensitive than the aerobic bottle. The three methods were positive for all inocula, even the lowest (viable inoculum below 10 CFU/ml for each fungus). Contamination was detected within 24 hours by the aerobic bottles in 91% (40/44), by Sabouraud in 98% (43/44) (no significant difference) and by visual inspection in 66% of cases (29/44) (p<0.001 with the two others). Maximum times to detection were 46, 48 and 72 hours respectively. CONCLUSION: This study further counters the preconception that fungal contamination is hard to detect in corneal organ culture media. This study is the last step in validating the use of a pair of blood bottles for the sterility testing of organ culture media, this time for fungi. Their use should make it possible to shorten microbiological quarantine and thus deliver corneas with higher endothelial cell density, without increasing the risk of recipient contamination.     

Attachment culture of human retinoblastoma cells: long-term culture conditions and effects of dibutyryl cyclic AMP

Cells from the Y-79 human retinoblastoma line were successfully grown as long-term monolayer cultures in serum-supplemented or serum-free, defined medium after pretreatment of the substrate with poly-D-lysine and fibronectin. A significant proportion of the cells differentiated morphologically in serum-free, defined medium as well as in serum-containing medium after treatment with dibutyryl cyclic AMP (dbcAMP). In the defined medium, approximately 30% of the cells showed development of long, ramifying processes, consistent with putative neuronal differentiation. Addition of dbcAMP in the defined medium resulted in an increasing number of differentiating cells (up to 50%); scattered flat cells were observed as well. Treatment of serum-supported cultures with dbcAMP resulted in a larger proportion (10%) of flat cells. Attempts to differentiate the cells with retinoic acid were unsuccessful. These results show that Y-79 cells can grow in attachment culture and differentiate into at least two distinct morphological cell types as well as offering an interesting system for studying the factors controlling growth and differentiation in human tumor cells in vitro.     

Use of a pair of blood culture bottles for sterility testing of corneal organ culture media

AIMS: To test the effectiveness and rapidity of a pair of blood culture bottles in the diagnosis of bacterial and fungal contamination of corneal organ culture media. METHODS: 761 microbiological analyses of storage media (Inosol and Exosol, Opsia, Toulouse, France), sampled in all phases of the organ culture at 31 degrees C of 410 consecutive corneas, were analysed. Each medium was inoculated in a pair of Bactec Plus Aerobic/F and Bactec Lytic/10 Anaerobic/F blood bottles and placed in a Bactec 9240 incubator for 14 days at 37 degrees C and in a Sabouraud broth at 20 degrees C. Changes in colour or turbidity of storage media were evaluated daily at the corneal bank. Recipients were screened post-graft for infectious signs. RESULTS: Overall contamination rate was 2.4% (18/761). Contamination was detected in less than 1 day in 78% (14/18) and less than 2 days in 94% (17/18). Positivity of the microbiological controls of starting media preceded changes medium colour in 10 out of 14 cases. Bactec blood bottles allowed detection of bacteria as well as yeasts. CONCLUSION: The use of a pair of Bactec blood culture bottles appears reliable for the rapid diagnosis of a wide range of microbiological contaminations of organ cultured corneas during banking.     

人眼视神经乳头星形胶质细胞体外培养:Ⅰ.原代培养和传代试验

目的:探索人眼视神经乳头星形胶质细胞体外培养的方法,为进一步研究星形胶质细胞在青光眼性视神经病变中的作用打下基础。方法:取材新鲜人眼视神经乳头组织和筛板组织,进行星形胶质细胞和筛板细胞的体外培养与传代试验。结果:组织块培养4~8wk后,原代细胞开始生长,星形胶质细胞在形态学和生长特性上与筛板细胞明显不同,β1型星形胶质细胞可以在无血清的培养液中生长良好,通过无血清培养液选择性培养可以在第二代传代过程中分离出纯化的星形胶质细胞,并可在第三至第四代收获大量细胞以备后续的研究。结论:精细准确的组织解剖分离对于获得纯化细胞至关重要,采用无血清培养液选择分离获得星形胶质细胞方法经济简单,细胞纯度高,便于进一步储存和研究。