Cultured corneal epithelia for ocular surface disease.
Cultured corneal epithelia for ocular surface disease.
复制标题
培养角膜上皮用于眼表疾病。
DOI：
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发表时间：
1999
期刊：
影响因子：
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通讯作者：
I. Schwab
中科院分区：
文献类型：
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作者：
I. Schwab
目的
评估来自体外培养的角膜缘角膜上皮干细胞的自体和同种异体扩增角膜上皮细胞移植治疗眼表疾病的潜在功效。
方法
人类受试者。在 19 名人类受试者中，有 18 名（20 次手术）使用各种上皮细胞载体进行了体外培养的角膜上皮细胞移植，以确定最有效的方法。 16 名患者（17 只眼睛进行了 18 次手术）接受了自体移植，2 名患者（每人 1 次手术）接受了同种异体移植。推测来自 1 名患者的角膜上皮干细胞在体外没有生长。扩增的角膜上皮细胞的载体包括角膜基质、1型胶原蛋白（Vitrogen）、软性隐形眼镜、胶原蛋白罩、自体移植物的羊膜和同种异体移植物的仅羊膜。对选定的供体移植物进行组织学确认。以羊膜为载体。进一步的研究以确定羊膜是否可能是扩张的角膜上皮细胞的最佳载体。研究人员对胰蛋白酶消化、超声处理、刮擦和洗涤的 17 种不同组合进行了研究，以找到去除羊膜上皮同时仍保留羊膜基底膜组织学外观的最简单、最有效的方法。假定的角膜上皮干细胞被收获并在体外扩增并应用于羊膜以创建复合移植物。因此，复合移植物由羊膜和扩张的角膜上皮干细胞组成，其中原始上皮已被去除，且基底膜没有明显的组织学损伤，而扩张的角膜上皮干细胞已应用于并成功粘附到裸露的羊膜上。动物模型。 12只兔子的1只眼睛的眼表以标准方式受损，直接去除假定的角膜缘干细胞、角膜上皮和相关上皮，随后应用正庚醇60秒。 6周后，所有受损的眼睛都上皮化并血管化。未经进一步处理而收获两只经过如此处理的眼睛，作为受损对照用于组织学研究。其余 10 只兔子接受了以标准方式应用于眼表的复合移植物（由羊膜和扩张的同种异体兔角膜上皮细胞移植物组成），然后戴上隐形眼镜。移植后第 16 天，处死 5 只兔子并取出角膜边缘用于组织学研究。第28天，处死剩余的兔子，并收获先前受损的眼睛用于组织学和免疫组织化学研究。
结果
人类受试者。在纳入该研究的 19 名患者中，1 名患者的假定角膜上皮干细胞在体外没有生长。其余 18 例患者（20 例手术，19 只眼）中，3 例患者结果不成功（3 例自体手术），1 例患者部分成功（同种异体手术），1 例患者目前结果未确定（同种异体手术）。一名不成功的患者患有睑内翻/倒睫，并机械切除了移植物，最终患上肺结核。另外 2 名不成功的患者推测自体供体上皮缺失，眼表疾病（翼状胬肉）复发。接受同种异体移植的部分成功患者患有感染性角膜炎，其上皮细胞再生延迟；他的视力只有很小的改善，但已经重新上皮化。接受第二次同种异体移植的患者在第4天失去了供体上皮。重新应用了额外的供体上皮，但目前结果尚未确定。以羊膜为载体。角膜上皮干细胞移植覆盖羊膜体外制备成功。组织学记录了羊膜上皮的去除和角膜上皮细胞的重新应用。动物模型。标准眼表损伤后未进行修复手术的两只兔子的组织学和免疫病理学符合不完全角膜上皮干细胞衰竭，伴有眼表血管化和疤痕。光学显微镜和 AE5 免疫组织学染色证实了眼表修复的结膜表型，但也记录了不完整的模型。同种异体干细胞移植有不同的结果。一只兔子出现化脓性感染并失去了移植物。其中 2 只兔子修复手术失败，3 只兔子部分失败，另外 3 只兔子部分成功，1 只兔子在 28 天时成功。组织学和免疫病理学研究记录了角膜上皮在受体表面的成功生长。
结论
1. 推测角膜上皮干细胞可以从角膜缘安全采集并在体外成功扩增。 2.扩增的角膜上皮细胞培养物可以生长在各种载体上，但目前剥脱的羊膜似乎是眼表修复的最佳载体。 3. 扩大的角膜上皮细胞移植似乎成功地重塑了受损的眼表，但需要细胞追踪和进一步确认。 4. 扩大同种异体角膜上皮细胞移植在技术上是可行的，并且可能代表针对特定眼表问题的替代治疗方式。 5.这些技术可能提供一种恢复正常眼表的新方法，同时最大限度地减少对侧或同级角膜缘角膜上皮干细胞的损伤或耗竭的威胁。 6. 兔子模型可能不完整，应谨慎解释。从任何眼睛中完全根除所有角膜上皮干细胞都很困难，这使得确认此类工作具有挑战性。 7. 兔模型的结果表明，同种异体移植物可以使某些眼表损伤恢复几乎正常的眼上皮表面。
PURPOSE
To evaluate the potential efficacy for autologous and allogeneic expanded corneal epithelial cell transplants derived from harvested limbal corneal epithelial stem cells cultured in vitro for the management of ocular surface disease.
METHODS
Human Subjects. Of the 19 human subjects included, 18 (20 procedures) underwent in vitro cultured corneal epithelial cell transplants using various carriers for the epithelial cells to determine the most efficacious approach. Sixteen patients (18 procedures on 17 eyes) received autologous transplants, and 2 patients (1 procedure each) received allogeneic sibling grafts. The presumed corneal epithelial stem cells from 1 patient did not grow in vitro. The carriers for the expanded corneal epithelial cells included corneal stroma, type 1 collagen (Vitrogen), soft contact lenses, collagen shields, and amniotic membrane for the autologous grafts and only amniotic membrane for the allogeneic sibling grafts. Histologic confirmation was reviewed on selected donor grafts. Amniotic membrane as carrier. Further studies were made to determine whether amniotic membrane might be the best carrier for the expanding corneal epithelial cells. Seventeen different combinations of tryspinization, sonication, scraping, and washing were studied to find the simplest, most effective method for removing the amniotic epithelium while still preserving the histologic appearance of the basement membrane of the amnion. Presumed corneal epithelial stem cells were harvested and expanded in vitro and applied to the amniotic membrane to create a composite graft. Thus, the composite graft consisted of the amniotic membrane from which the original epithelium had been removed without significant histologic damage to the basement membrane, and the expanded corneal epithelial stem cells, which had been applied to and had successfully adhered to the denuded amniotic membrane. Animal model. Twelve rabbits had the ocular surface of 1 eye damaged in a standard manner with direct removal of the presumed limbal stem cells, corneal epithelium, and related epithelium, followed by the application of n-heptanol for 60 seconds. After 6 weeks, all damaged eyes were epithelialized and vascularized. Two such treated eyes were harvested without further treatment, to be used for histologic study as damaged controls. The remaining 10 rabbits received composite grafts (consisting of amniotic membrane with expanded allogeneic rabbit corneal epithelial cell transplants) applied to the ocular surface in a standard manner followed by the application of a contact lens. At 16 days following transplantation, 5 of the rabbits were sacrificed and the corneal rims were removed for histologic study. At 28 days, the remaining rabbits were sacrificed and the previously damaged eyes were harvested for histologic and immunohistochemical study.
RESULTS
Human subjects. Of the 19 total patients admitted to the study, the presumed corneal epithelial stem cells of 1 patient did not grow in vitro. Of the remaining 18 patients (20 procedures, 19 eyes), 3 patients had unsuccessful results (3 autologous procedures), 1 patient had a partially successful procedure (allogeneic procedure), and 1 patient had a procedure with an undetermined result at present (allogeneic procedure). One unsuccessful patient had entropion/trichiasis and mechanically removed the graft and eventually went into phthisis. The other 2 unsuccessful patients suffered presumed loss of autologous donor epithelium and recurrence of the ocular surface disease (pterygium). The partially successful patient receiving an allogeneic transplant had infectious keratitis delay of his re-epithelialization; he has only minimal visual improvement but has re-epithelialized. The patient receiving the second allogeneic graft lost his donor epithelium at day 4. Additional donor epithelium was reapplied, but the result is undetermined at present. Amniotic membrane as carrier. The in vitro preparation of the amniotic membrane with corneal epithelial stem cell graft overlay was successful. Histology documented removal of the amniotic epithelium and reapplication of corneal epithelial cells. Animal model. The 2 rabbits that had no reparative surgery following standard ocular surface injury had histology and immunopathology consistent with incomplete corneal epithelial stem cell failure with vascularization and scarring of the ocular surface. Light microscopy and immunohistologic staining with AE5 confirmed the conjunctival phenotype of the ocular surface repair but also documented the incomplete model. The allogeneic stern cell transplants had varying results. One rabbit had a suppurative infection and lost the graft. Reparative surgery failed in 2 of the rabbits, failed partially in 3 of the rabbits, was partially successful in 3 others, and was successful in 1 rabbit at 28 days. Histologic and immunopathologic study documented successful growth of corneal epithelium onto the recipient surface.
CONCLUSIONS
1. Presumed corneal epithelial stem cells can be harvested safely from the limbus and expanded successfully in vitro. 2. Expanded corneal epithelial cell cultures can be grown onto various carriers, but currently denuded amniotic membrane seems to be the best carrier for ocular surface repair. 3. Expanded corneal epithelial cell transplants appear to resurface damaged ocular surfaces successfully, but cellular tracking and further confirmation are required. 4. Expanded allogeneic corneal epithelial cell transplants are technically possible and may represent alternative treatment modalities for selected ocular surface problems. 5. These techniques potentially offer a new method of restoring a normal ocular surface while minimizing the threat of damage or depletion to the contralateral or sibling limbal corneal epithelial stem cells. 6. The rabbit model was probably incomplete and should be interpreted with caution. The complete eradication of all corneal epithelial stem cells from any eye is difficult, making confirmation of such work challenging. 7. The results of the rabbit model suggest that allogeneic grafts may restore a nearly normal ocular epithelial surface to certain ocular surface injuries.