Ocular Surface Research Group, Singapore Eye Research Institute, Singapore.
Exp Cell Res. 2013 Nov 1;319(18):2781-9. doi: 10.1016/j.yexcr.2013.08.022. Epub 2013 Sep 5.
Pterygium is a fibrovascular growth on the ocular surface with corneal tissue destruction, matrix degradation and varying extents of chronic inflammation. To facilitate investigation of pterygium etiology, we immortalized pterygium fibroblast cells and profiled their global transcript levels compared to primary cultured cells.
Fibroblast cells were cultured from surgically excised pterygium tissue using the explant method and propagated to passage number 2-4. We hypothesized that intervention with 3 critical molecular intermediates may be necessary to propage these cells. Primary fibroblast cells were immortalized sequentially by a retroviral construct containing the human telomerase reverse transcriptase gene and another retroviral expression vector expressing p53/p16 shRNAs. Primary and immortalized fibroblast cells were evaluated for differences in global gene transcript levels using an Agilent Genechip microarray.
Light microscopic morphology of immortalized cells was similar to primary pterygium fibroblast at passage 2-4. Telomerase reverse transcriptase was expressed, and p53 and p16 levels were reduced in immortalized pterygium fibroblast cells. There were 3308 significantly dysregulated genes showing at least 2 fold changes in transcript levels between immortalized and primary cultured cells (2005 genes were up-regulated and 1303 genes were down-regulated). Overall, 13.58% (95% CI: 13.08-14.10) of transcripts in immortalized cells were differentially expressed by at least 2 folds compared to primary cells.
Pterygium primary fibroblast cells were successfully immortalized to at least passage 11. Although a variety of genes are differentially expressed between immortalized and primary cells, only genes related to cell cycle are significantly changed, suggesting that the immortalized cells may be used as an in vitro model for pterygium pathology.
翼状胬肉是一种眼表纤维血管生长，伴有角膜组织破坏、基质降解和不同程度的慢性炎症。为了便于翼状胬肉病因的研究，我们将翼状胬肉成纤维细胞永生化，并与原代培养细胞进行了全局转录水平的比较分析。
采用组织块培养法从手术切除的翼状胬肉组织中培养成纤维细胞，并传代至 2-4 代。我们假设干预 3 个关键分子中间产物可能是使这些细胞增殖所必需的。原代成纤维细胞通过含有人端粒酶逆转录酶基因的逆转录病毒构建体和另一个表达 p53/p16 shRNA 的逆转录病毒表达载体，依次被永生化。使用安捷伦基因芯片微阵列评估原代和永生化成纤维细胞之间的全局基因转录水平差异。
永生化细胞的光镜形态与传代 2-4 代的原代翼状胬肉成纤维细胞相似。永生化翼状胬肉成纤维细胞中表达了端粒酶逆转录酶，p53 和 p16 水平降低。在永生化和原代培养细胞之间，有 3308 个基因的转录水平发生了至少 2 倍的显著失调（2005 个基因上调，1303 个基因下调）。总的来说，与原代细胞相比，永生化细胞中有 13.58%（95%CI：13.08-14.10）的转录物的表达水平至少相差 2 倍。
翼状胬肉原代成纤维细胞成功永生化至至少第 11 代。虽然永生化和原代细胞之间有多种基因表达差异，但只有与细胞周期相关的基因发生了显著变化，这表明永生化细胞可用作翼状胬肉病理学的体外模型。
