体外培养重建角膜组织的技术探讨

目的：探讨体外重建角膜组织的三维培养技术。方法：原代培养角膜上皮,基质和内皮细胞,利用硫酸软骨素,透明质酸钠,I型胶原蛋白和微孔材料,建立三维培养系统,体外重建角膜组织结构,观察非醛交联,醛交联和微孔材料等三类人工角膜的透明度,常规病理组织学检测细胞在三维培养系统中生长状态。结果：非醛交联类重建人工角膜质地较软,透明度较低,光镜可见纤维结构,醛交联类重建人工角膜质地较韧,透明性较高,光镜可见均质结构,非吸收微孔材料类细胞长人较少,较难体外重建人工角膜,结论：利用生物工程技术能够体外重建人工角膜,非醛交联类结构较规则,醛交联类透明度较高。     

应用三维胶原凝胶培养法进行角膜重建的研究

以胶原凝胶为底物，可共同培养角膜上皮细胞、基质细胞及内皮细胞。上皮层通过空气－液体交界面培养，基质层及内皮层通过浸泡培养，重建三维结构的角膜组织模型。培养重建的角膜模型与生理条件的角膜组织结构相近，可应用于多种角膜组织的基础研究以及角膜移植术。还可以研究重建角膜模型在活体内的变化情况。     

应用三维胶原凝胶培养法进行角膜重建的研究

以胶原凝胶为底物，可共同培养角膜上皮细胞、基质细胞及内皮细胞。上皮层通过空气－液体交界面培养，基质层及内皮层通过浸培养，重三维结构的角膜组织模型。     

脱水保存角膜基质为载体培养角膜内皮细胞的实验研究

目的探讨以脱水保存角膜基质/后弹力层为载体培养角膜内皮细胞,构建组织工程化角膜内皮细胞移植膜的可行性及其机理。设计实验性研究。研究对象体外培养的兔角膜内皮细胞和脱水保存角膜基质/后弹力层。方法兔角膜经中性蛋白酶37°C孵育5min,去除内皮细胞保留后弹力层和角膜基质,无水氯化钙脱水后低温保存,使用前磷酸盐缓冲液复水。纯化的角膜内皮细胞接种于基质载体的后弹力层上进行体外培养,直至生长融合为细胞单层,倒置显微镜下观察细胞形态学变化。在不同时间点(1、2、4、6d)收集植片进行HE染色和电镜检测,分析组织结构的变化。主要指标角膜内皮细胞在脱水保存角膜基质/后弹力层载体上形成单层时间和生长特性,组织工程化角膜内皮细胞移植膜的三维结构和超微结构。结果角膜内皮细胞在载体上快速贴壁生长并增殖,体外培养6～7d即融合成单层,复合角膜内皮组织由基质/后弹力层和单层扁平内皮细胞组成,与生理状态下的角膜内皮组织相近。电镜下组织培养的兔角膜内皮细胞间连接紧密,细胞为多边形,胞核清晰,具有正常兔角膜内皮细胞的超微结构。结论角膜内皮细胞能够在干燥脱水保存基质/后弹力层载体上良好生长,并形成形态结构与正常角膜内皮组织相似的细胞单层,为角膜内皮细胞移植提供了新的载体选择。(眼科,2006,15:164-168)     

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。     

组织工程技术构建角膜基质层的实验研究

目的探索组织工程技术构建角膜的可行性,并为其提供构建角膜的理论依据和参数.方法应用组织工程方法构建角膜基质层.对兔角膜组织行体外分离获得角膜基质细胞,经培养扩增后接种于聚羟基乙酸生物支架上,然后将角膜基质细胞-生物材料复合物植入裸鼠皮下,6周后将植入物取出,行免疫组织化学染色及胶原纤维图像分析.结果光镜下,角膜基质细胞-生物材料复合物在裸鼠皮下所形成的新生组织呈网状板层结构.免疫组化染色新生组织Ⅰ型胶原表达阳性;胶原纤维图像分析,新生组织胶原纤维直径(27.7±6.20)nm与正常角膜胶原纤维直径(28.5±3.52)nm基质层相近.结论组织工程方法所构建的角膜基质组织已具备角膜基质层的组织学特征.     

转谷氨酰胺酶交联胶原凝胶构建三维角膜基质

目的检测转谷氨酰胺酶交联胶原凝胶对三维培养的角膜基质细胞的影响,探讨可提高机械性能的组织工程角膜基质层新途径。方法胶原酶消化法获取原代兔角膜基质细胞,以加入转谷氨酰胺酶与胶原凝胶交联为实验组,不加酶交联为对照组。倒置显微镜下每日观察细胞生长情况、Alamar-Blue试剂检测细胞增生、免疫荧光法检测凝胶内细胞波形蛋白、检测透光度、酶消化法检测胶原凝胶抗消化能力。结果实验组细胞胶原凝胶内附着和生长优于对照组,细胞在凝胶内呈树枝状生长。2组细胞均随培养时间延长明显增生（P=0.000）。共焦显微镜下见2组细胞胞浆波形蛋白均阳性表达,实验组细胞伪足更丰富。实验组透光度稍差于对照组。实验组抵抗胶原酶消化的能力显著增强。结论酶交联的胶原凝胶对角膜基质细胞无毒性作用,重构的基质层结构更加稳定,有利于组织工程角膜基质层的构建。     

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)     

体外构建含细胞组织工程角膜基质的方法

目的:研究体外构建组织工程角膜基质的方法.方法:用去垢剂联合酶的方法脱细胞处理猪角膜基质,在去细胞猪角膜基质材料上接种体外培养的兔角膜基质细胞,并将兔角膜基质细胞进行PKH26荧光标记,在体外构建组织工程角膜基质.用倒置荧光显微镜;HE染色光学显微镜;扫描电镜观察.结果:利用异种去细胞角膜基质组织为支架材料而构建的组织工程角膜基质具有近似正常角膜基质的三维立体结构.结论:以去细胞猪角膜基质材料为支架,利用组织工程技术可成功的在体外构建出含细胞的组织工程角膜基质组织.     

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。     

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.     

The effect of photodynamic action on corneal cells in tissue culture.

The effect of photodynamic inactivation on corneal epithelial, stromal and endothelial cells was investigated in tissue cultures. In contrast to reports regarding in vivo studies, the in vitro treatment caused significant degeneration and growth inhibition of stromal corneal cells and less marked changes of epithelial corneal cells in tissue cultures. Stationary endothelial cells appeared least effected. The neutral red was seen in the intracytoplasmic granules and in time localized mostly in the perinuclear area, and to a lesser extent within the nucleus. The in vitro study of actively growing corneal cells appears to be a sensitive indicator of cell damage following photoinactivation.     

In vitro transfer of rabbit corneal epithelium from carriers to denuded corneas or cryolathed lenticules.

Rabbit corneal basal epithelial cells seeded onto fixed gelatin membranes or commercial collagen shields formed 3 to 5 cell layers after 1 to 3 weeks of culture at 35 degrees C in nutrient medium. The cells grew better, by comparison, in the collagen shields and eventually formed a multilayered tissue that resembled the stratified morphology of native epithelium. Transfer of multilayered cultures (prior to stratification) from these carriers in vitro to denuded corneal buttons or cryolathed lenticules resulted in complete adhesion of the grafted tissue to the underlying recipient buttons after 24- to 48-h incubations. After mechanically removing the carriers, most of the epithelial cells remained attached to the stromal surface. Our experimental findings indicated that both kinds of carriers may be suitable for epithelial transplantation, although the collagen shield is probably superior because of its better biocompatibility and physical characteristics.     

Maspin: synthesis by human cornea and regulation of in vitro stromal cell adhesion to extracellular matrix.

PURPOSE: Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express maspin and whether maspin affects corneal stromal cell adhesion to extracellular matrix molecules. METHODS: Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant maspin was used to study attachment of cultured human corneal stromal cells to extracellular matrices. RESULTS: Maspin mRNA and micromolar amounts of the protein were found in all three layers of the human cornea in situ, including the stroma. Maspin was also detected in primary and first-passage corneal stromal cells, but its expression was downregulated in subsequent passages. Late-passage stromal cells, which did not produce maspin, responded to exogenous recombinant maspin as measured by increased cell adhesion not only to fibronectin, similar to mammary gland tumor epithelial cells, but also to type I collagen, type IV collagen, and laminin. CONCLUSIONS: The corneal stromal cell is the first nonepithelial cell type shown to synthesize maspin. Loss of maspin expression in late-passage corneal stromal cells in culture and their biological response to exogenous maspin suggests a role for maspin on the stromal cells in the cornea. Maspin may function within the cornea to regulate cell adhesion to extracellular matrix molecules and perhaps to regulate the migration of activated fibroblasts during corneal stromal wound healing.     

以异种角膜脱细胞基质为载体构建角膜上皮-载体-内皮复合物的实验研究

目的探讨以异种角膜脱细胞基质为载体,体外构建生物角膜的可能性和方法。方法应用去垢剂1％TritonX-100冷冻干燥处理制备猪角膜脱细胞基质载体,在其上皮面和内皮面分别接种兔角膜上皮细胞和内皮细胞,体外培养2周。将复合物制成组织切片,在光镜下观察组织形态（HE染色）,采用免疫组织化学方法检测角膜上皮特异性细胞角蛋白3（CK3）,使用锥虫蓝联合茜素红染色观察内皮细胞,在扫描电镜下观察上皮面和内皮面的超微结构。结果体外培养生物角膜获得上皮、无细胞基质和内皮三层复合结构。4或5层复层上皮中以扁平细胞为主,胞质内特异性CK3表达阳性;内皮层为一连续的单层扁平细胞,细胞活性良好,锥虫蓝联合茜素红染色显示组织呈典型的蜂巢样镶嵌结构。扫描电镜下观察,在载体的上皮面细胞呈复层样生长,形态近似扁平与梭形之间;内皮面为多边形单层细胞,表面具有微绒毛结构。结论构建的生物角膜为上皮一脱细胞基质载体一内皮复合物,初具角膜的雏形结构。异种角膜脱细胞基质提供了良好的细胞生长界面,有望成为体外构建角膜的载体材料。     

Iridoschisis associated with glaucoma and bullous keratopathy

Two of six patients (four women and two men ranging in age from 51 to 90 years) with bilateral senile iridoschisis also had secondary unilateral narrow angle or angle-closure glaucoma. All six patients had splitting of the iris stroma inferiorly. Histopathologic studies disclosed marked iris stromal atrophy without evidence of vascular or neural alterations. Vessels were present in the separated anterior stroma. Electron microscopy of iridectomy specimens from three patients and a corneal button from one disclosed scant or absent collagen fibrils in the area of separation. The cornea showed focal loss of endothelial cells, patchy posterior banding (110 nm) of Descemet's membrane, and stromal and epithelial edema. The posterior corneal surface showed degenerated endothelial cells and irregular connective tissue most marked inferiorly; the endothelial cells appeared to be normal.     

Growing human corneal epithelium on collagen shield and subsequent transfer to denuded cornea in vitro.

Three fundamental in vitro experiments have been done in the present report: 1) comparison of three different nutrient media on their abilities to culture and passage the human corneal epithelial cells; 2) evaluation of the ability of extracellular matrix material to promote the growth of cultured human corneal epithelium on collagen corneal shields; and 3) determination of the feasibility of the shield to serve as a carrier for the transfer of cultured cells to allogeneic, denuded corneal surface in vitro. Primary cultures of human corneal epithelium were established from explants which were obtained from limbal and peripheral corneal tissue by three different nutrient media respectively: KGM (Keratinocyte Growth Medium), SHEM (Supplemental Hormonal Epithelial Medium), and one combination of the two media (KGM/SHEM). We found the KGM/SHEM combination to be more favorable because morphology was better preserved, the proliferation rate increased five-fold over the 14 days observed time course, and we were able to subculture the tissue for at least three passages. With this combined medium, a suspension of cultured corneal epithelial cells (5 x 10(5)/ml) was seeded onto either the concave surface of collagen corneal shields or onto shields which had been coated with extracellular matrix materials (Matrigel or type IV collagen). The cells attached readily to all the coated shields (20/20) but to only a few of the uncoated shields (3/10), and formed a stratified tissue (2 to 3 layers) within seven days once the cells attached. However, the cells on the shields coated with Matrigel failed to become confluent under these conditions. The stratified tissue on type IV collagen coated shields could then be subsequently transferred to denuded human corneal stroma in organ culture by placing them together and incubating for 2-7 days. After that, histologic examinations showed that the epithelial cells had attached tightly to the recipient stromal surface, even after the removal of the collagen shield.     

圆锥角膜中细胞凋亡及Fas-L蛋白的表达

目的 探讨凋亡与圆锥角膜发病的关系及凋亡相关蛋白Fas-L的表达.方法 对20例圆锥角膜及5例正常角膜用原位末端标记法(TUNEL)检测凋亡,用免疫组织化学SP法检测Fas-L蛋白的表达;透射电镜观察凋亡细胞的形态学变化.结果 TUNEL染色示圆锥角膜组中上皮层、基质层及内皮层中细胞凋亡与正常角膜组比较差异均有统计学意义(P＜0.05);免疫组织化学示圆锥角膜组与正常角膜组基质层间Fas-L表达比较差异有统计学意义(P＜0.05);透射电镜可见圆锥角膜中存在凋亡特征的细胞.结论 圆锥角膜中存在凋亡,Fas-L蛋白的表达存在异常,Fas-FasL系统可能在圆锥角膜细胞凋亡中发挥了重要作用.