整合素α6β4及ECM配体在口腔黏膜钉突形态发生中的作用

The role of the integrin α6β4 and ECM ligands in the oral mucosa rete ridge morphogenesis Epithelium easily detached from scaffold or dermis is the technical bottleneck problem in mucosa tissue engineering. The deficiency of rete ridge is the mainly difference between mucosa and its engineered substitutes. Rete ridge enlarges contact area between epithelium and dermis for their adherence and permeating nutrition, provide an excellent microenvironment for the epidermal stem cells, maintain their self-renewal and long-term stabilization of epithelium. Therefore, to explore the mechanism of the rete ridge formation might provide a new approach for solving this problem. Rete ridges developed well with strong adhesion between the epithelium and the basal layer.This phenomenon provoked a question those structures for adhesion maybe one of the elements of that promote morphogenesis of epithelial rete ridges? Integrin α6β4 plays a leading role in the adhesion thus to be tested at first, the chronergy in integrin-mediated promotion and regulation to the rete ridge formation of receptor-specific mechanisms; following to investigate the synergy of the ECM ligands to the growth of rete ridges; finally to test of the surface morphology of the matrix material and the coating, whether the formation of rete ridge really relied on the surface conformation of dermal matrix. By cell cultivation, immunohistochemistry, biomarker, to observe the effects of integrin α6β4 and ECM ligands which promote natural rete ridge formation in the rete ridge rich region of the mucosa; to improve and fabricate a new acellular dermal matrix with rete ridge-like structure and cytokines, so that keratinocytes could easily anchor, proliferate, migrate, and undergo the morphogenesis of the rete ridge in such a microenvironment; to fabricate an oral mucosa equivalent with rich, elongated rete ridges.

黏膜上皮易与载体基质或真皮脱离是工程化黏膜制备的技术瓶颈，而人工黏膜在外形上与自然的区别在于缺乏上皮钉突样结构。钉突不仅增加上皮与基质之间的粘附面积，增大上皮营养供给的表面积；而且还具有维持干细胞生存微环境、持续自我更新与长期稳态的作用。 钉突发育较好的部位上皮与基底层粘附力较强，问题是起粘附作用的结构是否也就是促进上皮钉突形态发生的要素之一？因而首先测试在粘附中起主导作用的整合素α6β4；其次是ECM配体对钉突形成的协同作用；最后试验基质材料的表面形态和涂层对钉突的形成的诱导作用。 通过细胞培养、免疫组化和分子生物学细胞标记等技术，观察角化细胞生长发育成钉突的过程；以及整合素α6β4和ECM配体对钉突形成的诱导作用；根据ECM不同成份对角化细胞粘附生长的影响，改进并制备新的真皮基质，内含粘附因子外具凸凹结构，使黏膜种子细胞在易宜形成钉突的微环境中生长；从而制备出具备钉突形态的口腔黏膜替代品。

黏膜上皮易与载体基质或真皮脱离是工程化黏膜制备的技术瓶颈，而人工黏膜在外形上与自然的区别在于缺乏上皮钉突样结构。钉突不仅增加上皮与基质间的黏附面积，增大上皮营养供给的表面积；而且还具有维持干细胞生存微环境，持续自我更新与长期稳态的作用。本项目研究了整合素α6β4及ECM配体在口腔粘膜钉突形态发生中的作用。在体外实验中，用角化细胞生长因子（KGF）处理人永生化口腔上皮细胞（HIOEC），发现KGF促进HIOEC粘附和迁移且整合素α6β4表达量增高，同时使用整合素抑制剂HYD-1处理细胞发现细胞的黏附作用受到抑制且迁移能力减弱且整合素α6β4表达量降低；在动物模型中，KGF可促进口腔黏膜上皮钉突延长且整合素α6β4表达量增高，同时使用KGF与HYD-1局部注射，发现口腔黏膜上皮钉突并未发生明显变化。另外，本项目研究发现，I型胶原蛋白（Col I），III型胶原蛋白（Col III），IV型胶原蛋白（Col IV），纤连蛋白（FN），转化生长因子β（TGF-β）和结缔组织生长因子（CCN2）等纤维化相关分子在狗口腔粘膜中的表达与人类相似程度最高，提示其作为口腔粘膜纤维化研究动物模型的组织学的适宜性；同时通过探索包括角化，基底膜外观，上皮厚度，钉突长度，邻近钉突距离和局部变化的6种形态学特征和包括Ki67（增殖标记）和细胞角蛋白19（CK19;干性标记）的2种免疫组织化学标记来评估组织学特征表明：狗的粘膜与人类粘膜最相似，提示其作为动物模型的组织学基础。