脱细胞猪角膜基质的生物相容性与组织工程化兔角膜上皮移植的研究

目的 探讨脱细胞猪角膜基质的生物相容性,评价组织工程化角膜上皮组织作供体的可行性,观察支架材料的细胞化情况和种子细胞的存活情况.方法 实验研究.采用完全随机化设计的方法,用Dispase-Triton-X-100处理猪角膜基质,脱去角膜细胞;以角膜基质囊袋内植入的方法,观察异种角膜基质植入后的生物相容性,A组:脱细胞猪角膜基质,B组:新鲜猪角膜基质,C组:空白对照组.以组织工程化雄性角膜上皮组织为供体,同种雌性为受体,作板层角膜移植,观察角膜的混浊、水肿、新生血管等情况;组织病理学和免疫组化方法检测支架材料的细胞化情况,Y染色体性别决定基因(SRY)-聚合酶链反应(PCR)方法追踪种子细胞的存活情况.结果 猪角膜基质植入兔角膜囊袋后,角膜逐渐恢复透明,排斥反应指数＜6,组织病理学观察角膜结构完整,胶原纤维平行排列,少许细胞长入脱细胞猪角膜基质边缘,各组免疫组化检测未见CIM+、CD8+T淋巴细胞浸润.组织工程化角膜上皮作异体板层角膜移植后,3～4 d上皮光滑,10～20 d变为透明;15 d时角膜上皮、基质、内皮完整,上皮细胞约4或5层结构,少许基质细胞长入支架,1个月时可见角膜上皮细胞约7或8层细胞,基质纤维排列规则,多量细胞长入脱细胞角膜基质.上皮细胞表达CK3,支架内新生细胞表达波形蛋白.SRY-PCR结果显示种子细胞可以在受体内长期存活.结论 脱细胞猪角膜基质生物相容性良好,组织工程化角膜上皮可作为板层角膜移植的供体,脱细胞猪角膜基质细胞化良好,种子细胞可以在受体内长期存活.

Biocompatibility of acellular corneal stroma and transplantation of tissue-engineered corneal epithelium

Objective To evaluate the biocompatibility of xenogeneic acellular corneal stroma,the feasibility of tissue engineered corneal epithelial transplantation,and verify the long term survival of epithelial allograft Methods It was a experimental study.Porcine corneal stroma was treated by dispase followed by Triton-X-100 detergent.Treated porcine corneal stroma(group A)or fresh corneal stroma(group B)were put into the sac of rabbit cornea.Rabbit cornea without implantation of porcine corneal stroma was used as the control group(group C).Immunological rejection was evaluated in morphology,histopathology and immunohistochemistry in month 1,3,6,Female rabbit underwent Iamellar keratoplasty(LK)using male tissue engineered corneal epithelium as donors,and immunological reiection after LK Was analyzed.The comeas were collected at day 1,3,7,15 and 30 after LK and evaluated by histopathology,immunohistochemistry and sex-determining region of Y-chromosome(SRY)-PCR analysis.Results All comeas became transparent gradually after the transplantation of treated porcine corneal stroma and were not rejected.The epithelium,stroma,endothelium,Bowman's and Descemet's membrane were preserved in all corneas of group A and B in histological observations,collagen fibers were parallel,a few keratocytes presented in the acellular and fresh corneal stromas.The corneas of group C were normal in histological sections.No significant immune rciection was noted in any of the corneas.The corneas in the studv of transplantation of tissue engneercd cornea epithelium recovered smoothly in 3 or 4 days,turned transparent in 15 or 20 days after surgery and were not stained by fluorescein.Well-differentiated corneal epithelium were recognized at 15 and 30 days after LK.Many keratocytes infiltrated into the scaffold.SRY-PCR analysis showed that allogenic donor corneal epithelium cells could survive in recipients after a long-term observation.Conclusions Acollular porcine corneal stroma shows a satisfying biocompatibility.Tissue engineered corneal epithelium using acellular porcine corneal stronm as carrier could be used as donors for LK with satisfactory results.Donor cells have the potential to survive in recipients after long-term observation.