以自体结膜成纤维细胞为饲养细胞构建组织工程兔角膜上皮
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摘要
目的:创建一种新的组织工程角膜上皮构建系统,以自体结膜成纤维细胞为饲养细胞,避免异种饲养细胞的介入,用于治疗角膜缘干细胞缺乏性疾病。
方法:以组织块法体外培养新西兰白兔结膜成纤维细胞。使用不同浓度丝裂霉素C 37℃下处理培养的新西兰白兔自体结膜成纤维细胞(第1-2代)2小时,以MTT比色法检测丝裂霉素C的毒性,观察安全剂量范围内丝裂霉素C处理对细胞增殖的抑制作用,选取最适浓度处理兔结膜成纤维细胞制作饲养细胞。取兔颊部口腔粘膜上皮组织,以人去上皮羊膜上为载体,DMEM:F12(3:1)为基础培养液,构建组织工程角膜上皮(n=8)。观察兔口腔黏膜上皮细胞的生长、增殖、分化状况,并对构建的组织工程角膜上皮进行组织病理学和电子显微镜检查。
结果:兔结膜成纤维细胞可耐受浓度≤25μg/ml丝裂霉素C37℃处理2小时。制作饲养细胞的最适合处理浓度为5μg/ml。兔口腔粘膜上皮细胞于2-4天内从组织块周边爬出,8-10天左右即可接近融合。融合的细胞呈现典型的上皮样细胞形态,细胞大小均一,排列紧密,边界清晰,呈现铺路石样。以兔结膜成纤维细胞为饲养细胞构建的组织工程角膜上皮组织形态与正常兔角膜上皮相似,呈现为4-5层分化良好的复层上皮,基底部细胞呈矮柱状,基底上层是2-3层的翼状细胞,表层细胞扁平。电子显微镜检查显示构建的组织工程角膜上皮的显微结构亦与正常兔角膜上皮接近,包括细胞间以大量桥粒连接,基底细胞通过许多半桥粒牢固连接于羊膜以及表层上皮表面有微绒毛样结构。
结论:以兔自体结膜成纤维细胞代替3T3成纤维细胞作为饲养细胞可成功构建大体与显微结构均与正常兔角膜上皮相似的组织工程角膜上皮。
PURPOSE. To report the feasibility of using rabbit autologous conjunctival fibroblasts instead of 3T3 fibroblasts as feeder cells for the cultivation of transplantable oral mucosal epithelial cell sheets.
METHODS. Rabbit conjunctival fibroblasts were harvested using explant culture method. The safety dosage of mitomycin C for rabbit conjunctival fibroblasts was analyzed using MTT method. Appropriate dose was selected within the safety range to suppress the proliferation of rabbit conjunctival fibroblasts. Rabbit oral mucosal epithelial cells were cultured on denuded human amniotic membrane with autologcus conjunctival fibroblasts derived feeder cells. Tissue-engineered comeal epithelium was evaluated by HE stain, scanning and transmission electron microscope.
RESULTS. The safety dosage of mitomycin C to treat rabbit conjunctival fibroblast;; at 37℃for 2hours was under 25μg/ml. rabbit conjunctival fibroblasts cultured in 5μg/ml mitomycin C at 37℃for 2hours showed satisfying proliferation inhibition. Rabbit oral mucosal epithelial cells could be seen growing from the edges of the explants with 4 days, and reached confluence in 8-10 days. The cultivated oral epithelial sheet had four to five layers of stratified, well-differentiated cells. Electron microscope revealed that the epithelial cells were tightly connected with numerous desmosomal junctions, and were attached to the amniotic membrane with hemidesomoseomes. The apical surface of the cells were covered with microvilli.
CONCLUSIONS. Autologous rabbit conjunctival fibroblasts could be used as feeder cells for engineering corneal epithelium to avoid the use of potentially hazardous xenologic 3T3 fibroblasts.
方法:以组织块法体外培养新西兰白兔结膜成纤维细胞。使用不同浓度丝裂霉素C 37℃下处理培养的新西兰白兔自体结膜成纤维细胞(第1-2代)2小时,以MTT比色法检测丝裂霉素C的毒性,观察安全剂量范围内丝裂霉素C处理对细胞增殖的抑制作用,选取最适浓度处理兔结膜成纤维细胞制作饲养细胞。取兔颊部口腔粘膜上皮组织,以人去上皮羊膜上为载体,DMEM:F12(3:1)为基础培养液,构建组织工程角膜上皮(n=8)。观察兔口腔黏膜上皮细胞的生长、增殖、分化状况,并对构建的组织工程角膜上皮进行组织病理学和电子显微镜检查。
结果:兔结膜成纤维细胞可耐受浓度≤25μg/ml丝裂霉素C37℃处理2小时。制作饲养细胞的最适合处理浓度为5μg/ml。兔口腔粘膜上皮细胞于2-4天内从组织块周边爬出,8-10天左右即可接近融合。融合的细胞呈现典型的上皮样细胞形态,细胞大小均一,排列紧密,边界清晰,呈现铺路石样。以兔结膜成纤维细胞为饲养细胞构建的组织工程角膜上皮组织形态与正常兔角膜上皮相似,呈现为4-5层分化良好的复层上皮,基底部细胞呈矮柱状,基底上层是2-3层的翼状细胞,表层细胞扁平。电子显微镜检查显示构建的组织工程角膜上皮的显微结构亦与正常兔角膜上皮接近,包括细胞间以大量桥粒连接,基底细胞通过许多半桥粒牢固连接于羊膜以及表层上皮表面有微绒毛样结构。
结论:以兔自体结膜成纤维细胞代替3T3成纤维细胞作为饲养细胞可成功构建大体与显微结构均与正常兔角膜上皮相似的组织工程角膜上皮。
PURPOSE. To report the feasibility of using rabbit autologous conjunctival fibroblasts instead of 3T3 fibroblasts as feeder cells for the cultivation of transplantable oral mucosal epithelial cell sheets.
METHODS. Rabbit conjunctival fibroblasts were harvested using explant culture method. The safety dosage of mitomycin C for rabbit conjunctival fibroblasts was analyzed using MTT method. Appropriate dose was selected within the safety range to suppress the proliferation of rabbit conjunctival fibroblasts. Rabbit oral mucosal epithelial cells were cultured on denuded human amniotic membrane with autologcus conjunctival fibroblasts derived feeder cells. Tissue-engineered comeal epithelium was evaluated by HE stain, scanning and transmission electron microscope.
RESULTS. The safety dosage of mitomycin C to treat rabbit conjunctival fibroblast;; at 37℃for 2hours was under 25μg/ml. rabbit conjunctival fibroblasts cultured in 5μg/ml mitomycin C at 37℃for 2hours showed satisfying proliferation inhibition. Rabbit oral mucosal epithelial cells could be seen growing from the edges of the explants with 4 days, and reached confluence in 8-10 days. The cultivated oral epithelial sheet had four to five layers of stratified, well-differentiated cells. Electron microscope revealed that the epithelial cells were tightly connected with numerous desmosomal junctions, and were attached to the amniotic membrane with hemidesomoseomes. The apical surface of the cells were covered with microvilli.
CONCLUSIONS. Autologous rabbit conjunctival fibroblasts could be used as feeder cells for engineering corneal epithelium to avoid the use of potentially hazardous xenologic 3T3 fibroblasts.
引文
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