| The normal ocular surface is covered by corneal and conjunctival epithelia, each of which divides from their own stem cells and displays distinct programs of differentiation, and they are characterized by the expression of different pairs of keratins: Ck3/12 for the corneal epithelium and Ck4/13 for the conjunctival epithelium, together they maintain the ocular surface integrity. The corneal epithelium rests on a compact and avascular stroma, and the conjunctival epithelium on a vascular loose connective tissue. The mechanisms by which these varied ocular epithelial phenotypes established and maintained are unknown, but at least two hypotheses have been proposed: the different epithelial stem cells are important determinants of the distinct ocular epithelial phenotypes; however, we don't negate the possibility that distinct epithelial cell types give rise to the various regions and the stroma dictates the pattern of epithelial differentiation. In other words, it remains unclear whether growth and differentiation of these two epithelia are controlled by their respective stroma.Mesenchyme, which is defined as loose undifferentiated embryonic connective tissue, is critically involved in the development of the integument, urinary, gastrointestinal, skeletal and urogenital systems. The adult counterpart of the embryonic mesenchyme is stroma, which constitutes the non-epithelial component of an organ. The role of stromal-epithelial interactions in adulthood has received considerably less attention than embryonic mesenchymal-epithelial interactions. In part, this is because adult epithelial cells are thought to be irreversibly determined and terminally differentiated. But new findings have radically altered this concept. The stem cells dose not exclude the eventuality that there exists a hierarchy of progenitor cells that may revert back, may have differential plasticity and may not be restricted to one tissue type. Recently, experimental animal models have demonstrated mesenchyme-induced changes in the morphology, differentiation and growth of adult epithelial cells. These types of experiments challenge the concept that epithelial differentiation is irreversibly determined during embryonic and early postnatal life.Base on these earlier findings, we investigated the possible effects of corneal stroma on conjunctival epithelial cells. To test our hypothesis, we cultured adult rabbit conjunctival epithelial cells and implanted them homologically into rabbit corneal stroma. With proteinic and genic analyses, we found that the transplanted conjunctival epithelial cells were well grew and transdifferented to corneal epithelial phenomenon when confronted with corneal stroma. The base of this phenomenon was not clear, but our transplantation experiments showed clearly that signals from adult rabbit corneal stroma could be recognized by, and elicited transformation of adult rabbit conjunctival epithelial cells to corneal epithelium. In addition, and perhaps more significantly, the detailed chain of events provides greater insight into questions relating to stem cell lineages and cells reprogramming and it remains an exacting possibility for clinical application including organ reconstruction. The New Zealand rabbits were chosed as the experimental objects. Firstly, we will understand the proteinic expressions of the corneal and conjunctival epithelial cells and establish the foundation for the farther experiments.Cytokeratin is a set of framework protein of cells, which is formed by pairs of acidic keratin and basic keratin. These cytokeratins are regarded as the molecular markers of the type and differentiation of epithelial cells, and that the Ck3/Ck12 are designated as the corneal-type differentiated markers, whereas Ck4/Ck13 designated as the conjunctival-type differentiated markers. Ck5/Ck14 designated as the markers of basic layer cells. Connexin43 participates in the proliferation and differentiation of cells. It can be expressed normally in the corneal epithelial cells, but seldom appears in conjunctival epithelial cells, negative at limbal position. E-cadherin is the modulated factor of proliferation and subsistence in cells. It is important on the connecting between cells and expresses normally at eye lid and corneal epithelial cells. Muc5AC is the marker of mucin that is secreted by conjunctival goblet cells.Normally collagenIV which is synthesized by epithelial and mesenchymal cells consists of basement membrane. It modulates the proliferation and differentiation of cells. Laminin is also an important protein of basement membrane. Its dominating function is promoting the adhesive and movement of cells.In rabbit eyes, Ck3 and Ck12 were expressed in corneal epithelial cells, connexin43 and E-cadherin were normally express in corneal epithelial cells also. But these proteins expressed negatively at limbal position. Ck4, Ck13 as well as Muc5AC were expressed in conjunctival epithelial cells. Ck14 expressed at basal layer of epithelial cells normally. And CollagenIV and Laminin were expressed at the position between epithelial and stroma of ocular surface. According to these immunochemical staining results which were resemble with the epithelial cells of human ocular surface, we chosed the New Zealand rabbits as the objects for our experimental amnimal model.Chapter 2 The cultivation and identification of rabbit conjunctival epithelail cellsAccording to the plasticity of adult stem cells, we assume that we can pick up and culture the rabbit conjunctival epithelial (stem) cells, then induce them to differentiate to corneal epithelial cells, which can be used for ocular surface reconstruction. The purpose of this experiment is to culture and identify rabbit conjunctival epithelial cells, which is the foundation of next step for our experiments.Most researchers recognized that P63 was the marker of stem cells and expressed in all kinds of proliferous epithlelial cells. It is important in reproducting of stem cells. Cytokeratin is the marker of growth and differentiation of epithelial cells, and the subtype of cytokeratin is expressed according to the different stage of cytodifferention. Therefore, the subtype of cytokeratin is an important marker for identifying the origin of epithelial cells. Ck14 is the marker of proliferous epithelial cells, Ck4 and Ck13 are designed as the mature conjunctival epithelial cells. Muc5AC that is the marker of goblet cells indicates the differentiation and maturation of conjunctival epithelial cells also.Our experimental results showed that after one week's culture, one layer of epithelial cells was formed. After a series of immunochemical staining, most of cells were positive with P63 and Ck14, parts of cells were positive with Ck4, just a few cells expressed Muc5AC, but while Ck13 almost negative completely. These results proved that after one week's culture, the mixed members were included several differentiation stages of conjunctival epithelial cells, most of them were stem cells and transient amplify cells, but the differentiated cells were contained inside also.Chapter 3 The animal model of conjunctival epithelial cells trans- differentiation in the corneal stromaThe ASC transdifferentiation is induced by the micro environment, The corneal stroma is the micro environment of corneal epithelial. For inducing the conjunctival epithelial cells to transdifferentiate following the corneal direction in vivo, the cultured the conjunctival epithelial cells are regarded as the seeds, and the central corneal stroma as the inducing environment.The rationale for using adult rabbit central corneal stroma in vivo instead of corneal surface as a inducer is many folds: (1) Corneal and limbal stroma is in fact a mesenchyme which normally induces and supports the differentiation of both limbal and corneal epithelial cells. Cells grow in central stroma are easy to get stronger signal from fibroblasts. (2) Because the conjunctivalization of corneal surface in vivo is invariably accompanied by corneal vascularization and inflamation, these pathologic changes will bring in other unknown serum and tear factors, which may further complicate the entire modulating mechanism. Our central corneal stroma model in vivo will be avoided these infections in the process of induction. (3) It is difficult to study conjunctival transdifferentiation on ocular surface in vivo, because the source of migrating epithelial cells might be mixed by residual corneal epithelial cells. This contamination will be avoided by our central corneal stroma model also.We established an animal model of conjunctival epithelial cells transdifferentiated in corneal stroma in vivo successfully. Our experimental results validated that conjunctival epithelial cells survived well in corneal stroma.Chapter 4 The protein and gene expression of the transdifferentiation corneal-like epithelial cellsThe purpose of our experiments is to validate the possibility of the transdifferentiation of conjunctival cells in corneal stroma with the changes of genic and proteinic expressions.When adult rabbit cultured conjunctival epithelial cells encountered with itself corneal stroma, epithelial cytodifferentiation was radically changed step by step and such profound changes in epithelial cytodifferentiation were associated with the change of genic expression. Taking this experimental approach we showed immunostaining results here that within 3 days of being implanted, these cells arranged irregularly and a few Ck3/12 positive cells began to emerge from implanted conjunctival cells. After days between 7 to 14 days, the cultured cells began to form organization, whereas the Ck3/12 expression was up-regulated, but Ck4 expression still existed. According to Real-time PCR, the expression of Ck4 was detected in high level in normal rabbit conjunctival epithelial cells and rapidly decreased in conjunctival epithelial cells once they were placed in corneal stroma 3 days and onwards, but still exist. At the meantime, the expressions of Ck3/12 were deteced in samples as early as 3 days, and was observed up to 2 weeks. This finding allowed us to conclude that the phenotype of the epithelial cell differentiation in vivo was determined by both intrinsic divergence and external environment. In addition, the expressions of basement membrane such as collagenIV and laminin were from faintly and discontinuously to strongly and continuously between the implanted cells and corneal stroma. This approved that there was a"cross talk"between the implanted conjunctival epithelial cells and the contacted corneal stroma, this promoted the synthesis and precipitation of these two kinds of basement membranes.The results we give shows that conjunctival epithelial cells can be transdifferentiated with the inducing of corneal stroma. This is another powerful attestation for the transdifferentiation of adult stem cells. It indicates the potential foreground that the conjunctival stem cells are cultured as a new source for tissue engineer cornea. But the mechanism of transdifferentiation and the function of the transdifferentiated corneal epithelial cells phenotype are still unclear and need to be explored thoroughly in future. |
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