Stromal niche controls the plasticity of limbal and corneal epithelial differentiation in a rabbit model of recombined tissue
Stromal niche controls the plasticity of limbal and corneal epithelial differentiation in a rabbit model of recombined tissue
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DOI：
10.1167/iovs.03-0584
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发表时间：
2003-12-01
影响因子：
4.4
通讯作者：
Tseng, SCG
中科院分区：
文献类型：
--
作者：
Espana, EM;Kawakita, T;Tseng, SCG
目的.成年兔角膜缘基底上皮含有角膜上皮干细胞，其特征在于角蛋白-3（K3）的阴性表达和连接蛋白43（Cx43）的低表达。本研究旨在确定角膜缘基质是否决定角膜缘和角膜上皮分化的可塑性。通过将50 mg/mL分散酶II在补充激素上皮培养基（SHEM）中于4 ℃孵育18小时，从荷兰带状兔中分离中央角膜和色素利姆布斯的活上皮片。通过抗K3、Cx43、整合素β 1和胶原蛋白IV的抗体免疫染色研究切割平面。通过活-死测定评估源自这些片的单细胞的活力。将这样的角膜缘（L）和角膜（K）上皮片与角膜缘（Ls）或角膜（Ks）基质重组，并在SHEM中培养10天，然后提升到空气-流体界面1周。通过组织学和对K3和Cx43的免疫染色确定所得上皮表型，并通过Hoechst和TUNEL核染色研究细胞凋亡。分离的角膜缘和角膜上皮片的活力测定为91.1% +/-2.9%。分离的角膜缘上皮片的基底上皮对整合素β 1呈阳性，对K3呈阴性，但对Cx43呈弱阳性，并且仍然保留IV型胶原斑块。所有重组体均显示出复层上皮，仅在K/Ks中观察到上皮内囊肿和脱落碎片，以及L/Ls的插入过滤器上的上皮生长。K3在L/Ls基底层中表达阴性，而在K/Ks基底层中表达阳性。K3在L/Ks基底层呈零星阳性表达，而在K/Ls基底层则呈阴性表达。Cx43在K/Ks细胞基底层均匀表达，而在L/Ls、K/Ls和L/Ks细胞基底层表达较弱。凋亡指数仅在K/KS上皮内囊肿中增高。这些结果强烈表明，角膜缘基质调节上皮分化，增殖和凋亡的方向有利于干，而角膜基质促进分化。进一步调查构成这种利基的因素，应有助于揭开控制严厉的秘密。
PURPOSE. The adult rabbit limbal basal epithelium contains corneal epithelial stem cells, which have been characterized by a negative expression of keratin-3 (K3) and a lower expression of connexin 43 (Cx43). This study was conducted to determine whether the limbal stroma dictates the plasticity of limbal and corneal epithelial differentiation.METHODS. Viable epithelial sheets of the central cornea and the pigmented limbus were isolated from Dutch belted rabbits by incubation of 50 mg/mL of dispase II in supplemental hormonal epithelial medium (SHEM) for 18 hours at 4degreesC. The cleavage plane was studied by immunostaining with antibodies against K3, Cx43, integrin beta1, and Collagen IV. Viability of single cells derived from these sheets was assessed by a live-dead assay. Such limbal (L) and corneal (K) epithelial sheets were recombined with either limbal (Ls) or corneal (Ks) stroma, and cultured in SHEM for 10 days before lifting to the air-fluid interface for I week. The resultant epithelial phenotype was determined by histology and immunostaining to K3 and Cx43, and apoptosis was investigated by Hoechst and TUNEL nuclear staining.RESULTS. Viability of isolated limbal and corneal epithelial sheets was determined to be 91.1% +/- 2.9%. The basal epithelium of isolated limbal epithelial sheets was positive for integrin beta1, negative for K3, but weakly positive for Cx43, and still retained patches of Collagen IV. All recombinants showed stratified epithelia, with intraepithelial cysts with desquamated debris noted only in K/Ks, and epithelial outgrowth onto the insert filter from L/Ls. As expected, expression of K3 was negative in the basal layer of L/Ls, but positive in that of K/Ks. Expression of K3 was sporadically positive in the basal layer of L/Ks but largely negative in that of K/Ls. Expression of Cx43 was uniformly expressed in the basal layer of the K/Ks, but weak in that of L/Ls, K/Ls, and L/Ks. A higher apoptosis index was only noted in intraepithelial cysts of K/Ks.CONCLUSIONS. These results strongly indicate that the limbal stroma modulates epithelial differentiation, proliferation and apoptosis in the direction favoring sternness, whereas the corneal stroma promotes differentiation. Further investigation into elements constituting such a niche should help unveil the secrecy whereby sternness is controlled.