Mucin gene expression in immortalized human corneal-limbal and conjunctival epithelial cell lines
Mucin gene expression in immortalized human corneal-limbal and conjunctival epithelial cell lines
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DOI：
10.1167/iovs.02-0851
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发表时间：
2003-06-01
影响因子：
4.4
通讯作者：
Russo, CL
中科院分区：
文献类型：
--
作者：
Gipson, IK;Spurr-Michaud, S;Russo, CL
目的.覆盖眼表的角膜和结膜上皮细胞在防止病原体进入眼睛和通过为其顶表面产生高度亲水性粘蛋白分子来维持湿表面表型方面起重要作用。眼表感染、创伤和导致干眼症的病理学威胁视力并可导致失明。了解粘蛋白提供的眼表面防御机制受到了阻碍，因为缺乏永生化的人角膜和结膜上皮细胞系，这些细胞系保留了天然组织的粘蛋白基因表达模式。本研究的目的是利用粘蛋白基因表达作为分化标志物来表征新开发的永生化角膜和结膜细胞系。如先前公开的方法所述衍生细胞系。角膜缘和结膜上皮细胞的原代培养物被依次转导以表达显性负性P53蛋白和p16（INK 4A/Rb）抗性突变cdk 4蛋白，这使得细胞能够绕过最近鉴定的角质形成细胞原代培养物的衰老机制。然后这些细胞被转导以表达端粒酶的催化亚基，从而允许它们保留端粒并无限分裂。在塑料、I型胶原或基质胶上培养、与成纤维细胞共培养以及在严重联合免疫缺陷（SCID）小鼠中培养后，测定了两种细胞系（命名为HCLE的人角膜缘细胞系和HCjE的人结膜细胞系）中的细胞形态和粘蛋白基因表达。通过逆转录、实时聚合酶链反应、免疫印迹分析或免疫组化检测上皮细胞粘蛋白的表达，并与天然角膜和结膜中的表达进行比较。当生长在高钙培养基上的塑料和I型胶原蛋白，细胞的两个线分层，表现出多个细胞层。在基质胶中，两种细胞系均形成含有管腔的细胞聚集体。在SCID小鼠中，结膜细胞系在肾包膜下形成分层。角膜细胞系表达角蛋白K3和K12，角蛋白是角膜上皮特异性的，两种细胞系都表达K19。与天然组织一样，HCLE和HCjE细胞系表达膜相关粘蛋白MUC 1、MUC 4和MUC 16，尽管它们的水平通常较低。MUC 4和-16 mRNA的水平与天然组织最具可比性，特别是当在塑料上培养时。分层培养物的顶端细胞是表达膜相关粘蛋白MUC 1和MUC 16的细胞。杯状细胞特异性MUC 5AC的mRNA和蛋白质检测到一小群HCjE细胞时，仅使用I型胶原作为底物或当细胞与成纤维细胞共培养。两种细胞系均产生糖基化粘蛋白，如H185抗体（一种识别粘蛋白上的碳水化合物表位的抗体）的结合所示。永生化角膜（HCLE）和结膜（HCjE）细胞微粒表现出其天然上皮的粘蛋白基因表达谱。这些细胞系将用于确定眼表面粘蛋白基因表达的调节，并可能用于杯状细胞分化。（Invest Ophthalmol维斯科学。2003;44：2496-2506）DOI：10.1167/iovs.020851。
Purpose. The corneal and conjunctival epithelia, which cover the ocular surface, play an important role in preventing pathogen penetrance into the eye and maintaining a wet-surface phenotype by producing highly hydrophilic mucin molecules for their apical surfaces. Ocular surface infections, wounding, and pathologies resulting in dry eye threaten sight and can cause blindness. Understanding the ocular surface defense mechanisms that mucins provide has been hampered by the lack of immortalized human corneal and conjunctival epithelial cell lines that retain mucin gene expression patterns of the native tissue. The purpose of this work was to characterize newly developed immortalized corneal and conjunctival cell lines using mucin gene expression as markers of differentiation.Methods. The cell lines were derived as described by a previously published process. Primary cultures of corneal-limbal and conjunctival epithelia were sequentially transduced to express a dominant negative P53 protein and a p16(INK4A/Rb) resistant, mutant cdk4 protein, which enabled the cells to bypass a senescence mechanism recently identified for primary cultures of keratinocytes. These cells were then transduced to express the catalytic subunit of telomerase to permit them to retain their telomeres and divide indefinitely. Cellular morphology and expression of mucin genes in the two cell lines, designated HCLE for the human corneal-limbal line and HCjE for the human conjunctival cell fine, were determined after culture on plastic, type I collagen, or Matrigel, in coculture with fibroblasts, and in severe combined immunodeficient (SCID) mice. Expression of the epithelial cell mucins was assayed by reverse transcription, real-time polymerase chain reaction, immunoblot analysis, or immunohistochemistry and compared with expression in native cornea and conjunctiva.Results.. When grown in high-calcium medium on plastic and type I collagen, cells of both lines stratified, exhibiting multiple cell layers. In Matrigel, both cell lines formed cell aggregates that contained lumens. In the SCID mice, the conjunctival cell line formed stratified layers under the kidney capsule. The corneal cell line expressed keratins K3 and K12, the keratins that are corneal-epithelial-specific, and both cell lines expressed K19. As in native tissue, the HCLE and HCjE cell lines expressed the membrane-associated mucins, MUC1, -4, and -16, although their levels were generally lower. Levels of MUC4 and -16 mRNA were the most comparable to native tissue, particularly when cultured on plastic. Apical cells of the stratified cultures were the cells that expressed the membrane-associated mucins MUC1 and -16. Goblet-cell-specific MUC5AC mRNA and protein was detected in a small population of HCjE cells only when using type I collagen as a substrate or when cells were cocultured with fibroblasts. Both cell lines produced glycosylated mucins as indicated by binding of H185 antibody, an antibody that recognizes a carbohydrate epitope on mucins.Conclusions. The immortalized corneal (HCLE) and conjunctival (HCjE) cell fines exhibit the mucin gene expression repertoire of their native epithelia. These cell lines will be useful in determining regulation of ocular surface mucin gene expression and, potentially, goblet cell differentiation. (Invest Ophthalmol Vis Sci. 2003;44:2496-2506) DOI:10.1167/iovs.020851.