Characterization of putative stem cell phenotype in human limbal epithelia
Characterization of putative stem cell phenotype in human limbal epithelia
复制标题
DOI：
10.1634/stemcells.22-3-355
复制
发表时间：
2004-01-01
期刊：
影响因子：
5.2
通讯作者：
Li, DQ
中科院分区：
文献类型：
--
作者：
Chen, Z;De Paiva, CS;Li, DQ
本研究评估了与干细胞（SC）特性相关的分子标记物，旨在表征人角膜缘上皮中假定的SC表型。在垂直和水平子午线上切割人角膜和角膜缘组织进行组织学、透射电子显微镜（TEM）和免疫染色。半定量逆转录-聚合酶链反应（RT-PCR）和原位杂交用于评估基因表达。透射电镜显示角膜缘基底细胞为小型原始细胞。免疫组化显示p63、ABCG 2和整合素α 9主要表达于利姆布斯基底上皮细胞。针对整合素β 1、表皮生长因子受体（EGFR）、K19、烯醇化酶-α和CD 71的抗体使利姆布斯的基底细胞比基底上上皮更明亮。整合素α 6、巢蛋白、E-钙粘蛋白和连接蛋白43不染色角膜缘基底细胞，但角膜和利姆布斯的基底上上皮显示强免疫反应性。K3和总苞蛋白只对角膜和角膜缘浅层细胞染色。RT-PCR结果显示，角膜缘上皮中p63、ABCG 2和整合素α 9 mRNA的表达水平高于角膜上皮，而K3、K12和连接蛋白43的表达水平低于角膜上皮。原位杂交结果显示p63基因转录产物定位于角膜缘上皮基底层。本研究提示，利姆布斯基底上皮细胞p63、ABCG 2和整合素α 9阳性，巢蛋白、E-钙粘蛋白、连接蛋白43、外皮蛋白、K3和K12阴性，整合素β I、EGFR、K19和烯醇化酶α表达相对较高。这种假定的SC表型可能有助于角膜缘上皮SC的鉴定和分离。
This study evaluated proposed molecular markers related to stem cell (SC) properties with the intention of characterizing a putative SC phenotype in human limbal epithelia. Human corneal and limbal tissues were cut in the vertical and horizontal meridians for histology, transmission electron microscopy (TEM), and immunostaining. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization were used to evaluate gene expression. TEM showed that the limbal basal cells were small primitive cells. Immunostaining disclosed that p63, ABCG2 and integrin alpha9 were primarily expressed by the basal epithelial cells of limbus. Antibodies against integrin beta1, epidermal growth factor receptor (EGFR), K19, enolase-alpha, and CD71 stained the basal cells of the limbus more brightly than the suprabasal epithelia. Integrin alpha6, nestin, E-cadherin and connexin 43 did not stain the limbal basal cells, but the suprabasal epithelia of the cornea and limbus showed strong immunoreactivity. K3 and involucrin stained only corneal and limbal superficial cells. RT-PCR showed higher levels of p63, ABCG2 and integrin alpha9 mRNA, but lower levels of K3, K12 and connexin 43 expressed in the limbal epithelia than the corneal epithelia. In situ hybridization showed that p63 transcripts were located in basal layer of the limbal epithelium. This work suggests that the basal epithelial cells of the limbus are p63, ABCG2 and integrin alpha9 positive, and nestin, E-cadherin, connexin 43, involucrin, K3, and K12 negative, with relatively higher expression of integrin betaI, EGFR, K19, and enolase-alpha. This putative SC phenotype may facilitate the identification and isolation of limbal epithelial SCs.