不同培养方法对组织工程化小鼠角膜上皮构建影响的比较研究

目的:探讨滋养细胞在小鼠角膜上皮细胞复层化中的作用,并研究构建组织工程化角膜上皮的理想方法。方法:在Transwell气液界面培养系统中,分别采用接触滋养层培养法、分离滋养层培养法、复式滋养层培养法以及无滋养层培养法等4种方法进行组织工程化小鼠角膜上皮的重建。HE染色进行组织学观察,免疫荧光法检测p63、角蛋白19以及involucrin的表达。结果:接触滋养层培养法、分离滋养层培养法中,角膜上皮复层化为3-4层;复式滋养层培养法中,角膜上皮复层化达5-7层;而无滋养层培养法中,复层化仅为2-3层。复式滋养层培养法构建的小鼠角膜上皮,基底细胞层和基底细胞上层表达祖细胞标记p63和角蛋白19,角膜上皮全层均表达分化标记involucrin。结论:复式滋养层培养法为构建组织工程化小鼠角膜上皮的理想方法。

Comparative study of four culture methods to engineer murine corneal epithelial sheet

AIM:To investigate the roles of feeder cells ln stratification of murine corneal epithelial cells and build an ideal method to engineer stratified epithelial sheet.METHODS:Using contact feeder culture,separated feeder culture,compound feeder culture and culture without feeder cells by Air-lifting method in Transwell chamber culture system,tissue engineered corneal epithelium was reconstructed.Corneal sheets were stained with hematoxylin and eosin(HE)for histological observation.The expression of p63 and keratin 19(K19)and involucrin(IVL)was investigated by immunocytochemistry analysis.RESULTS:Stratification was limited to three to four layers in the contact feeder group,whereas separate feeder sheets were slightly more stratified.The compound feeder group produced a stratified epithelium with five to seven layers of cells.The group without 3T3 feeder cells formed only two to three layers of cells.Immunostaining images in the compound feeder group showed expression of progenitor markers p63 and K19 in the basal and suprabasal layer,as well as differentiation marker involucrin in all layers.CONCLUSION:The remarkable stratification as well as the Iimbal phenotype makes the compound feeder system a candidate tool for cultivating transplantable epithelial sheets.