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。     

猪脱细胞角膜基质组织结构特点与生物相容性研究

背景构建组织工程化角膜时,载体的选择十分重要。目前有多种载体可供选用,但脱细胞角膜基质是公认的较好载体。目的观察猪脱细胞角膜基质的组织结构特点,评价其与异种角膜基质和上皮细胞的生物相容性。方法取猪角膜组织进行组织块培养,经胰蛋白酶-EDTA酶消化角膜上皮、基质、内皮细胞,支架组织于-20℃冷冻干燥后灭菌保存。猪脱细胞角膜基质石蜡切片行常规苏木精一伊红染色,光学显微镜下观察组织的形态学特征;扫描电镜下观察其组织结构特点;并对其物理特性,如抗拉性、膨胀性、含水量和透明度进行评价。将猪脱细胞角膜基质移植到兔角膜基质层内,同时与体外培养的兔角膜上皮细胞共培养4周,分析其组织和细胞生物相容性。结果经酶消化处理后猪角膜组织中未见上皮、基质和内皮细胞,其胶原纤维直径大小、排列与正常角膜组织相同,抗拉性、膨胀性、含水量和透明度等物理特性与正常猪角膜相似。猪脱细胞角膜基质行异种兔角膜基质层间移植1周时可见轻度水肿,2周后水肿基本消退,4周时透明度较好。猪脱细胞角膜基质与兔角膜基质之间贴附良好,未见炎症反应及新生血管。兔角膜上皮细胞接种于猪脱细胞角膜基质上共培养4周后CK3表达阳性。猪脱细胞角膜基质脱水前与脱水2h、4h后及正常猪角膜基质间的抗拉性、膨胀性、含水量的差异均无统计学意义（P〉0．05）,但脱水2h、4h后及正常猪角膜基质的透明度明显好于脱水前,差异均有统计学意义（P〈0．01）。结论猪脱细胞角膜基质组织结构与正常猪角膜相似,与兔角膜基质和上皮细胞具有良好的生物相容性。     

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。     

脱细胞猪角膜基质体外支持角膜上皮和基质细胞的生长

目的:探讨脱细胞猪角膜基质体外能否支持兔角膜细胞的生长。方法:体外培养兔角膜上皮细胞和基质细胞,并接种到制备的脱细胞猪角膜基质上,倒置相差显微镜和组织学观察细胞生长情况。结果:上皮细胞能在脱细胞猪角膜基质上贴附生长,10d时可形成2~3层的复层结构。基质细胞在脱细胞猪角膜基质上贴附生长后可向材料深层迁徙。结论:制备的脱细胞猪角膜基质体外可支持兔角膜上皮细胞和基质细胞的生长。     

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     

层粘连蛋白在大鼠角膜碱烧伤中的表达及意义

目的　研究层粘连蛋白 (laminin ,LN)在大鼠角膜碱烧伤模型中表达的变化 ,揭示其在角膜碱烧伤修复过程中的作用。方法　制作大鼠角膜碱烧伤模型 ,以免疫组化的方法观察LN在烧伤后 12h、2 4h、3d、7d、14d时表达的变化。结果　LN在正常大鼠角膜仅在基底层柱状上皮细胞胞浆和胞间以及基底膜有少许表达 ;烧伤后 12h ,基底柱状上皮细胞胞浆和胞间以及基底膜的表达增多 ,同时角膜基质层、角膜固有细胞出现LN ,并于 3～ 7d达高峰 ;14d角膜修复时 ,在基底膜柱状细胞、基质中表达减少 ,在角膜基质中表达基本消失。结论　LN在角膜碱烧伤过程中扮演重要角色 ,在介导淋巴细胞吞噬坏死组织、介导细胞 细胞间、细胞 细胞外基质间的黏附、介导上皮细胞移行中有重要作用 ,对于角膜的重新上皮化有积极作用。     

EGF联合KGF促进人角膜上皮细胞生长

目的:寻找促进角膜上皮损伤修复,治疗持续性角膜上皮缺损的有效药物方法:用~3H—胸腺嘧啶核苷(~3H—TDR)掺入及液体闪烁技术,观察外源性表皮生长因子(EGF)联合角蛋白细胞生长因子(KGF)对体外培养的角膜上皮细胞DNA合成的影响,并计算细胞倍增时间。结果:10ng/mlEGF,10ng/mlKGF单独或联合应用均有明显促进人角膜上皮细胞DNA合成的作用(与对照组比较 P<0.01),联合用药,作用更强(P<0.05)。应用EGF与KGF明显缩短了细胞倍增时间。结论:外源性EGF与KGF对体外培养的人角膜上皮细胞有明显的促细胞增生作用,联合用药,效果更佳。表明EGF与KGF具有应用于临床,促进角膜上皮损伤修复的可能性。眼科学报1996; 12:107-109。     

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。     

表皮生长因子对兔角膜上皮细胞生长作用的实验研究

研究了表皮生长因子（ＥＧＦ）对角膜上皮细胞的生长影响，用氚标的胸腺嘧啶核甘（＾３ＨＴｄＲ）掺入法证明ＥＧＦ单独对角膜上皮细胞无生长促进作用，而当和角膜基质块同时培养时，ＥＧＦ能够明促进膜上细胞生长，不同浓度ＥＧＦ的每分钟放射性计数（ＣＰＭ）差异均具有显著性意义（Ｐ〈０．０５或Ｐ〈０．００１），并且随ＥＧＦ浓度增加对角增上皮细胞促生长作用增强。提示ＥＧＦ能促进角膜上皮细胞的生长作用，但需要角膜基质的     

角质细胞生长因子促人角膜上皮细胞生长的研究

目的寻找促进角膜上皮损伤修复的有效方法。方法用３Ｈ胸腺嘧啶核苷（３ＨＴｄＲ）掺入及液体闪烁技术，观察角质细胞生长因子（ＫＧＦ）对体外培养的人角膜上皮细胞ＤＮＡ合成的影响，并计算细胞倍增时间。结果１～１００ｎｇ／ｍｌＫＧＦ有明显促进人角膜上皮细胞ＤＮＡ合成的作用，且呈剂量依赖性（ｒ＝０．９２３３，Ｐ＜０．００１）。１０ｎｇ／ｍｌＫＧＦ明显缩短了细胞倍增时间（３１．５９±４．８８ｈ，与对照组４０．９８±５．２０ｈ比较，Ｐ＜０．０５）。结论外源性ＫＧＦ对体外培养的人角膜上皮细胞有明显的促细胞增生作用。表明ＫＧＦ具有应用于临床，促角膜上皮损伤修复的可能性。     

Construction of a complete rabbit cornea substitute using a fibrin-agarose scaffold

PURPOSE: To construct a full-thickness biological substitute of the rabbit cornea by tissue engineering. METHODS: Ten rabbit corneas were surgically excised, and the three main cell types of the cornea (epithelial, stromal, and endothelial cells) were cultured. Genetic profiling of the cultured cells was performed by RT-PCR for the genes COL8 and KRT12. To develop an organotypic rabbit cornea equivalent, we used a sequential culture technique on porous culture inserts. First, endothelial cells were seeded on the base of the inserts. Then, a stroma substitute made of cultured keratocytes entrapped in a gel of human fibrin and 0.1% agarose was developed. Finally, cultured corneal epithelial cells were grown on the surface of the scaffold. Stratification of the epithelial cell layer was promoted by using an air-liquid culture technique. Corneal substitutes were analyzed by light and electron microscopy. RESULTS: All three types of corneal cells were efficiently cultured in the laboratory, expanded, and used to construct a full-thickness cornea substitute. Gene expression analyses confirmed that cultured endothelial cells expressed the COL8 gene, whereas epithelial cells expressed KRT12. Microscopic evaluation of the cornea substitutes demonstrated that epithelial cells tended to form a normal stratified layer and that stromal keratocytes proliferated rapidly in the stromal substitute. The endothelial monolayer exhibited a pattern similar to a normal corneal endothelium. CONCLUSIONS: These findings suggest that development of a full-thickness rabbit cornea model is possible in the laboratory and may open new avenues for research.     

Protective effect of uridine on cornea in a rabbit dry eye model

PURPOSE: To investigate the effect of uridine on cultured human corneal epithelial cells and keratocytes in vitro and to evaluate whether the application of uridine-containing eye drops could improve ocular surface health in an in vivo dry eye model. METHODS: Uridine was added to cultured epithelial cells (3 x 10(4) cells/well) and keratocytes (1 x 10(4) cells/well) at various concentrations (0.5-50 microM). Cytotoxicity was tested with the use of MTT assay, and the cells were assessed for apoptosis with the use of flow cytometry. Expressions of hyaluronic acid (HA), glycosaminoglycan (GAG), nitric oxide (NO), and matrix metalloproteinase (MMP)-9 were measured. In vivo, the degree of reepithelialization was assessed after topical application of uridine (100 microM) in a rabbit corneal wound model. Changes in tear production and conjunctival goblet cell counts were investigated after instillation of various concentrations of uridine-containing eye drops in a rabbit dry eye model. RESULTS: In vitro, uridine showed no cellular toxicity. It increased the biosynthesis of HA and GAG and reduced MMP-9 levels in cultured corneal epithelial cells and keratocytes. In vivo, uridine enhanced corneal wound healing and significantly increased the number of conjunctival goblet cells in rabbits. CONCLUSIONS: Uridine can restore the health of the ocular surface in a rabbit corneal wound and dry eye model.     

Differential regulation of fibronectin synthesis in three different types of corneal cells

The production of fibronectin (FN) and its response to serum or epidermal growth factor (EGF) were investigated in three different types of rabbit corneal cells cultured in vitro. The corneal epithelial cells, stromal fibroblasts (keratocytes) and endothelial cells were separately cultured in different media: basic medium containing minimal serum (0.5%), basic medium with supplementary serum at a final concentration of 10% and basic medium with 100 ng/ml EGF, respectively. FN production by each type of cell was examined either by the immunofluorescent staining method or by the metabolic labeling method followed by immunoprecipitation of FN in the culture medium. Each type of corneal cell produced and secreted FN. FN secretion into the culture medium by keratocytes and by endothelial cells was enhanced by the addition of EGF. However, FN secretion by epithelial cells was lowered by the additional serum or EGF. Furthermore, when the epithelial cells were cultured in the basic medium, DNA synthesis was low but FN secretion was high. These results suggest that the control mechanism of FN production differs between epithelial cells and keratocytes or endothelial cells.     

Comparison of the bacteriostatic effects, corneal cytotoxicity, and the ability to seal corneal incisions among three different tissue adhesives

PURPOSE: To compare the bacteriostatic effects, corneal cytotoxicity, and ability to seal corneal incisions among fibrin glue and 2 commercially available cyanoacrylate derivatives: N-butyl cyanoacrylate and methoxypropyl cyanoacrylate. METHODS: The bacteriostatic activities of these tissue glues were verified by measuring the zones of bacterial growth inhibition surrounding the adhesive droplets on agar plates inoculated with Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli, or Mycobacterium chelonae. Corneal cytotoxicity was tested by a direct contact method by using cultured bovine corneal epithelial cells, keratocytes, and corneal endothelial cells challenged with droplets of adhesives. Each of the cells was treated with droplets of adhesives. The ability to seal corneal incisions was verified by calculating the maximum intraocular pressure resistant to leakage of rabbit corneal stab wounds sealed with tissue adhesives. RESULTS: Methoxypropyl cyanoacrylate and N-butyl cyanoacrylate showed bacteriostatic effects against S. aureus, S. pneumoniae, and M. chelonae but not P. aeruginosa and E. coli. In contrast, fibrin glue had no such effects against either Gram-positive or -negative bacteria (P < 0.01). Methoxypropyl cyanoacrylate showed the highest levels of corneal cytotoxicity, followed by N-butyl cyanoacrylate. Fibrin glue, however, showed minimal cytotoxicity (P < 0.01). Methoxypropyl cyanoacrylate and N-butyl cyanoacrylate also displayed a greater ability to seal corneal incisions than that of fibrin glue (P < 0.01). CONCLUSIONS: The bacteriostatic effects, corneal cytotoxicity, and ability to seal corneal incisions differed among the 3 compounds tested. These different properties should be considered when choosing tissue adhesives during corneal surgery.     

Tropoelastin biosynthesis by corneal cells. Epithelial inhibition of keratocyte tropoelastin biosynthesis

The vertebrate cornea is an avascular tissue and does not contain elastic fibers. We tested the capacity of corneal epithelial cells and stromal keratocytes to synthesize tropoelastin. Explant cultures and cell cultures were obtained from these two cell types in standard culture conditions. Their elastin-synthetic activity was compared to skin explant cultures and to dermal fibroblast cell cultures. Both corneal cell types synthesized tropoelastin as shown by the incorporation of a radioactive precursor followed by immunoprecipitation of tropoelastin. When serial cultures of keratocytes were tested, tropoelastin biosynthesis strongly increased after the 3rd passage and was at the 9th passage more than the double of that of the first passage. When cocultures were studied with or without cell contact, epithelial cells partially inhibited tropoelastin biosynthesis by keratocytes. This inhibition was somewhat stronger (-36%, p < 0.005) with cell-to-cell contact than keeping separate epithelial cells and keratocytes bathing in the same medium (-18%, p < 0.005). When human skin fibroblasts were substituted for keratocytes with cell-to-cell contact, their tropoelastin biosynthesis was also inhibited by corneal epithelial cells (-42%, p < 0.005), to the same extent as for keratocytes. In Transwell culture, this inhibition was again somewhat lower (-36%, p < 0.005). Some diffusible factor produced by epithelial cells is apparently involved. The epithelial inhibition of tropoelastin biosynthesis by stromal keratocytes might represent one of the mechanisms keeping corneal stroma exempt of elastin fibers.     

20%乙醇处理兔角膜后上皮增生和细胞凋亡的研究

目的探讨准分子激光角膜上皮瓣下磨镶术(LASEK)中采用20%乙醇浸润兔角膜40s后角膜上皮增生和角膜细胞凋亡情况与机械刮除角膜上皮后的异同。方法实验组42只新西兰大白兔,用直径为8mm的LASEK专用角膜上皮刀切割角膜上皮,20%的乙醇浸润单眼40s,机械刮除对侧眼中央8mm直径的角膜上皮,随机分7组,于术后0、4h,1、3、5、8、30d取材;6只兔眼为空白对照。角膜冰冻切片,行Ki67免疫组化检查和TUNEL检测,计数角膜中央前基质细胞。结果乙醇浸润后5d中央角膜上皮增生达峰值,术后1d周边角膜上皮增生达峰值;术后4h上皮刀口下方局限的角膜基质细胞TUNEL染色阳性,数量最多;各组角膜中央前基质细胞计数和空白对照比差异无统计学意义(P=0.68)。机械刮除角膜上皮后3d周边角膜上皮增生达峰值,其高于乙醇浸润后角膜上皮的增生峰值;术后4h可见大量中央前基质细胞TUNEL阳性;术后1d中央前基质细胞数量最少(P<0.05)。结论与机械刮除角膜上皮相比,20%乙醇浸润40s对角膜损伤轻,恢复快,乙醇浸润后的角膜上皮对基质细胞有保护作用。     

Distribution of epidermal growth factor (EGF) receptors in rabbit corneal epithelial cells, keratocytes and endothelial cells, and the changes induced by transforming growth factor-beta 1.

Three kinds of rabbit corneal cells (epithelial cells, keratocytes and endothelial cells) were cultured, and the growth promoting effects of epidermal growth factor (EGF) were examined in these cells. It was found that the sensitivity of the epithelial cells to EGF was the highest, that of the endothelial cells was a little lower, and that of the keratocytes was the lowest. Transforming growth factor-beta 1 (TGF-beta 1) did not influence growth of the three kinds of corneal cells. It was found that TGF-beta 1 enhanced the growth promoting effect of EGF in the keratocytes, but not in the epithelial and endothelial cells. EGF receptors in the corneal cells were labelled with [125I]EGF and analysed by Scatchard plot. In the epithelial and endothelial cells, both high and low affinity receptors were found, while the keratocytes had only low affinity receptor. In the epithelial cells, the number and the association constant of the high affinity receptors were, respectively, 2.79 x 10(4) per cell and 0.034 nM, and those of the low affinity receptors were, respectively, 13.4 x 10(4) per cell and 0.700 nM. In the endothelial cells, the number and the association constant of the high affinity receptors were 1.27 x 10(4) per cell and 0.086 nM, respectively, and those of the low affinity receptors were 8.91 x 10(4) per cell and 1.536 nM. In the keratocytes, the number of receptors and the association constant were 9.49 x 10(4) per cell and 1.535 nM, respectively. The corneal cells were treated with TGF-beta 1 for 24 hr and its influence on EGF receptors was examined. The results showed that TGF-beta 1 induced the high affinity receptors in the keratocytes, although TGF-beta 1 did not influence EGF receptors in the epithelial and endothelial cells. The number of high affinity receptors in keratocytes treated with TGF-beta 1 was 1.02 x 10(4) per cell and the association constant was 0.171 nM (this was approximately tenfold higher than that of the receptor of keratocytes not treated with TGF-beta 1).     

Growth of acanthamoeba on human corneal epithelial cells and keratocytes in vitro

Acanthamoebic keratitis, a potentially devastating infection usually associated with contact lens wear, has been recognized with increasing frequency in recent years. Once the Acanthamoeba organisms gain access to the human cornea, it is not clear which constituents of the corneal milieu provide a substrate for their growth. The growth of Acanthamoeba polyphaga was investigated on cultured monolayers of human corneal epithelial cells, stromal keratocytes, and stromal homogenate suspensions. Growth was determined through organism counts and observation of cytopathic effects on tissue culture dishes. Compared with tissue culture media controls, acanthamoebic growth was supported by cultured epithelial cells and keratocytes but not stromal homogenates. These results suggest that in acanthamoebic keratitis the organisms depend on the cellular components of the cornea as substrates for growth. This in vitro model may also provide further information on the pathogenesis of keratitis and a system for drug sensitivity testing.     

胎儿角膜上皮细胞分离方法的研究

目的:为了利用流产儿角膜组织体外培养获取角膜上皮细胞,做为构建组织工程化角膜上皮的种子细胞。方法:收集30例(60个角膜)人流产胎儿,分别采用酶消化法、酶消化法结合组织块法和组织块法3种方法分离并培养人胎儿角膜上皮细胞。结果:用Dispase和胰蛋白酶冷消化角膜上皮后,在获取细胞总数、细胞活力和原代培养成功率上没有明显差异,但用Dispase消化后原代细胞容易成活,传代可得到纯化的上皮细胞;酶消化法结合组织块法培养时加入角膜缘组织块可以缩短细胞形成单层的时间,增加传代后细胞的活力;组织块法培养时,在胎儿全角膜组织培养时可得到角膜上皮细胞,角膜组织切割后培养不能获得纯化的角膜上皮细胞。结论:体外构建组织工程化角膜上皮时,种子细胞的获取可以采用流产儿角膜组织为材料分离并培养角膜上皮细胞。