羊膜和角膜干细胞移植治疗眼表疾病临床观察

目的探讨羊膜移植、自体角膜干细胞移植及自体体外培养角膜干细胞移植重建眼表治疗眼表疾病的疗效。方法眼表疾病43例46眼（瘢痕性结膜囊狭窄、睑球粘连6例6眼，角结膜化学、热烧伤10例13眼，原发和复发性翼状胬肉切除术后结膜缺损27例27眼）进行羊膜移植、自体角膜干细胞移植及自体体外培养角膜干细胞移植，观察植片及角结膜上皮愈合、组织浸润和新生血管情况。结果羊膜移植术重建结膜囊效果满意，睑球粘连松解。羊膜、自体体外培养角膜干细胞移植术治疗角结膜烧伤均可使眼表上皮化，基质炎性浸润减轻，新生血管明显减少；自体体外培养角膜干细胞移植对角膜缘干细胞损伤严重的患者效果显著。自体和自体体外培养角膜干细胞移植治疗翼状胬肉均有良好效果，术后不适症状轻时间短，角膜创面愈合快，随访（1～2）y两种术式各复发1眼。自体体外培养角膜干细胞移植对复发性翼状胬肉切除术后较大的结膜缺损更为适合。结论羊膜移植、自体角膜干细胞移植和自体体外培养角膜干细胞移植对重建眼表都有重要的功效，手术方式的选择必须根据角膜干细胞损伤的程度、范围进行选择。     

显微羊膜移植技术重建严重眼表烧伤临床观察

目的 评价显微羊膜移植技术重建眼表烧伤的临床效果。方法 对22例（24眼）严重眼表烧伤患者分别采用单纯羊膜移植术，羊膜移植联合异体新鲜角膜缘移植术，羊膜移植结膜囊成形术，并随访观察术后疗效，结果 13例（15例）单纯羊膜移植患者中，11例（17眼）无角膜结膜进行性溶解和穿孔，无新生血管和假性胬肉侵入角膜表面，2例因眼睑缺损，眼表干燥导致羊膜，角膜溶解穿孔，眼球萎缩，羊膜联合异体角膜缘移植术5例中，眼表结构恢复正常。无假性胬肉及睑球粘连发生，羊膜移植结膜囊成形术4例中，3例恢复了眼球的运动功能，1例因植片下积血，预后较差，再次发生眼球粘连，结论 运用显微手术羊膜移植重建眼表烧伤，可有效减少角膜溃疡穿孔，预防或修复睑球粘连，减少眼表新生血管，羊膜被认为是眼表重建较为理想的生物膜。     

羊膜移植术治疗兔重度角结膜碱烧伤后遗血管性角膜白斑

目的探讨羊膜移植术在重建眼表面、治疗重度角结膜碱烧伤后遗血管性角膜白斑的作用。方法采用甘油保存的人羊膜移植术治疗兔重度角结膜碱烧伤后遗血管性角膜白斑１０例（１０眼）。结果４例羊膜上皮面朝上者，术后１５～２０ｄ角膜表面上皮被覆完整，３０ｄ病理显示结膜上皮向角膜上皮转化；５例朝下和１例羊膜水肿者，术后羊膜被破坏，眼表面重建失败。结论羊膜移植术可用于重度角结膜碱烧伤晚期眼表面重建，支持羊膜无抗原性、有助于角膜上皮形成的观点     

羊膜移植术治疗严重眼表碱烧伤

目的 观察羊膜移植术对治疗严重眼表碱烧伤的临床疗效。方法 对10例严重角膜碱烧伤患者，根据不同病情施行新鲜羊膜和保存羊膜材料移植治疗，并通过观察患者术后视功能情况及眼表的完整性来评价疗效。结果 羊膜移植术重建活动期眼表碱烧伤可促进眼表上皮快速愈合，防止持续性上皮缺损和角膜基质坏死发生。抑制新生血管形成，避免睑球粘连。结论 羊膜移植术对治疗严重的眼表碱烧伤有较好的疗效。     

新鲜羊膜与保存羊膜移植治疗严重眼表疾病疗效比较

目的比较新鲜羊膜与保存羊膜移植治疗严重眼表疾病的效果。方法84例84眼随机分为新鲜羊膜移植组39眼与甘油保存羊膜移植组45眼,术后观察其临床疗效、并发症。结果角膜病中视力提高者新鲜组16眼、保存组18眼,无新生血管生长者新鲜组14眼、保存组16眼,羊膜早期溶解者新鲜组4眼和保存组7眼,分别进行统计学处理均无显著性差异(P>0.05),角膜、结膜上皮化愈合均在术后3～4周完成。随访3～10个月,所有病例均获稳定眼表。结论在眼表修复与重建过程中新鲜羊膜与保存羊膜的疗效、预后基本一致。     

羊膜移植术在严重眼部碱烧伤中的临床应用

目的 评价羊膜移植术治疗严重眼部碱烧伤的效果。方法 采用羊膜移植术治疗严重眼部碱烧伤23例29眼。结果 29眼中25眼重建成功，术后羊膜固定好，角膜局部炎症减轻，角结膜逐渐愈合。4眼术后4～10天内出现羊膜脱落、融解。结论 羊膜移植术可以使碱烧伤的角结膜创面迅速上皮化，控制角膜的进一步环死和融解，减少新生血管的形成，但对角膜水肿、浑浊严重的病例，羊膜移植术的效果欠佳。     

谈羊膜移植在角膜和眼表疾病应用中的问题

由于羊膜具有抗炎、抗纤维化、抗新生血管生成、促进角膜修复的作用,羊膜已经成为治疗角膜和眼表疾病的理想材料.随着羊膜广泛应用于角膜和眼表疾病,在某种程度上改变了一些角膜及眼表疾病传统的治疗方式,但随之而来的临床问题也逐渐显露,主要体现在：适应证如何把握;手术如何进行个体化设计;如何观察术后并发症和术后规范用药等问题.在此,谈些个人的经验和看法,以期使羊膜移植手术能在临床治疗角膜和眼表疾病中发挥更好的作用.     

羊膜移植在眼表疾病的临床应用

目的 探讨和评估新鲜羊膜移植治疗眼表疾病的疗效.方法 对结膜肿物,睑球粘连,碱烧伤,酸烧伤,热灼伤共24例(28眼)施行新鲜羊膜移植术,术后观察眼表重建情况,羊膜植片情况,术后视力情况,随访4-18个月.结果 24例(28眼)均未发生新鲜羊膜植片的排斥反应.2 5眼一次羊膜移植术后结膜、角膜上皮化痊愈,眼表重建成功.睑球粘连的患者第三眼位复视消失.1眼发生睑球粘连,2眼角膜部分上皮缺损,再次行羊膜覆盖术,术后随访完成上皮化,术后视力不同程度提高.结论 新鲜羊膜移植能有效的促进上皮细胞的分化移行、抑制局部的炎症反应、阻止新生血管和瘢痕的形成、阻止烧伤的病理发展,是治疗眼表疾病的一种积极有效的治疗方法.     

重度眼表碱烧伤早期羊膜移植治疗的研究

目的 探讨新鲜羊膜早期移植在重度眼表碱烧伤的治疗效果.方法 采用新鲜羊膜早期移植治疗重度眼表碱烧伤35例38眼.结果 术后所有患眼眼表迅速稳定,20眼角膜恢复透明,10眼形成角膜白斑,4眼全角膜浑浊,3个月后24眼脱盲,视力≥0.2者11眼.结论 重度眼表碱烧伤早期进行新鲜羊膜移植可促进角膜上皮修复、抑制角膜新生血管侵入,防止睑球粘连.     

新鲜人羊膜移植在重建眼表结构中的临床应用

目的：评价新鲜羊膜移植手术在眼表结构重建中的临床效果。方法：对76例不同眼表疾病采用新鲜人羊膜移植手术以重建眼表结构，其中2例眼球碱烧伤角膜缘干细胞严重缺乏角膜溃疡，8例为大泡性角膜炎，4例为真菌性角膜炎，1例严重睑球粘连，61例翼状胬肉（4例为复发性）。结果：术后均未见新鲜羊膜植片急性排斥反应。碱烧伤的2眼，无角结膜进行性溶解和穿孔，无新生血管或假性胬肉侵入角膜表面。大泡性角膜炎，症状均消失；真菌性角膜炎，溃疡全部痊愈。61例翼状胬肉4例复发。结论：新鲜羊膜移植重建角结膜表面，减轻了炎症反应，减少新生血管的生成，抑制纤维组织增生。充分清除眼表的病变组织和羊膜移植片的缝合固定，对羊膜移植重建眼表结构，恢复功能极其重要。     

新鲜羊膜移植治疗早期重症眼部烧伤

目的 探讨新鲜羊膜移植联合结膜、角膜移植对早期重症眼部烧伤的治疗效果。方法 急性重症眼部烧伤患者21例22眼，分别行羊膜移植联合结膜移植12例12眼和羊膜、结膜移植联合板层或穿透性角膜移植9例10眼。术后观察羊膜、结膜及角膜植片情况，长后随访2～16个月，平均7个月。结果 19眼羊膜植片愈合良好；20眼穹隆部结膜植片全部存活；羊膜移植联合结膜移植中10眼术后未见角膜融解，角膜缘均未见新生血管长入。羊膜、结膜移植联合板层或穿透性角膜移植，1例2眼出现轻度排斥反应，另8眼角膜植片均恢复透明。结论 新鲜羊膜移植可有效地促进上皮细胞分化移行、抑制局部的炎症反应、阻止新生血管和瘢痕形成、阻止烧伤的病理发展减轻和推迟角膜植片的排斥反应。     

新鲜羊膜移植治疗难治性眼表疾病的临床观察

目的探讨新鲜羊膜移植治疗难治性眼表疾病的疗效。方法选择本院酸碱热烧伤5例、角膜溃疡10例、角膜上皮完全剥脱1例、大面积睑球粘连5例、结膜延迟愈合1例患者,共计22例(22只眼);采用单层或多层羊膜移植,术后随访观察6-12个月,平均9个月。结果 22例术后临床上均未见新鲜羊膜植片急性排斥反应;同时眼表迅速稳定。严重酸碱热烧伤的5例无角结膜进行性溶解和穿孔,甚至1例铁水烫伤伴角膜穿孔患者术后保全眼球;10例角膜溃疡患者,9例溃病愈合,视力不同程度提高,1例溃病复发(糖尿病患者),放弃再移植;5 例睑球粘连患者眼球运动功能恢复。结论新鲜羊膜移植是重建眼表的有效方法,它可以减轻角膜的炎症反应, 阻止眼表进行性溶解,加速眼表的稳定,同时减少角膜新生血管的形成。     

深低温保存羊膜治疗严重睑球粘连的临床观察

目的:探讨深低温保存羊膜移植治疗严重睑球粘连的临床价值。方法:对43例46眼有严重睑球粘连及假性胬肉的患者,进行睑球粘连松解后,应用羊膜移植重建球结膜和角膜表面,观察其临床效果。结果:在随访期内(平均13±3.5mo),24例患者疗效为优,眼睑球粘连均得到明显改善,眼表基本恢复正常;15例疗效为良,睑球粘连部分复发,但较术前有所改善;7例因上下睑球全粘连、结膜囊闭锁愈后效果较差。结论:对于严重的睑球粘连,深低温保存羊膜移植重建眼表是较理想的治疗方法。     

Sterilized, freeze-dried amniotic membrane: a useful substrate for ocular surface reconstruction

PURPOSE: To examine the feasibility of using sterilized, freeze-dried amniotic membrane (FD-AM) as a substrate for cultivating autologous corneal epithelial cells for ocular surface reconstruction. METHODS: Human AM deprived of amniotic epithelial cells by incubation with EDTA was freeze dried, vacuum packed, and sterilized with gamma-irradiation. The resultant FD-AM was characterized for its physical, biological, and morphologic properties by stretch stress tests, immunohistochemistry, electron microscopy, and cell culture. In addition, 3 weeks after an ocular surface injury, the conjunctivalized corneal surfaces of eyes in eight rabbits were surgically reconstructed by transplantation of autologous cultivated corneal epithelial cells on FD-AM. RESULTS: A stretch stress test revealed no significant differences between sterilized FD-AM and cryopreserved AM. Immunohistochemistry for several extracellular matrix molecules and electron microscopic analysis of FD-AM revealed that the process of drying and irradiation did not affect its biological and morphologic properties. The corneal epithelial cells cultivated on FD-AM had four to five stratified, well-differentiated cell layers. Corneas that were grafted with the cultivated corneal epithelial cells on FD-AM were clear and were all epithelialized at 10 days after surgery. CONCLUSIONS: The sterilized, freeze-dried AM retained most of the physical, biological, and morphologic characteristics of cryopreserved AM; consequently, it is a useful biomaterial for ocular surface reconstruction.     

Successful regrafting of cultivated corneal epithelium using amniotic membrane as a carrier in severe ocular surface disease

PURPOSE: Our group performed cultivated allogeneic corneal epithelial transplantation in 13 eyes from 11 patients with severe ocular surface disorders. After the clinical application of this new surgical treatment, some patients experienced epithelial and subepithelial opacities. We applied our procedure again in these patients to achieve successful ocular surface reconstruction. METHODS: The corneal limbal epithelial cells from donor corneas were cultivated for 4 weeks on denuded amniotic membrane (AM) carrier, with 3T3 fibroblast coculture and airlifting. The study subjects consisted of 3 patients. At 3 and 12 months after the first operation, the failed epithelial graft with AM was replaced with new allogeneic corneal epithelium cultivated on AM. RESULTS: At 48 hours after transplantation, the corneal surfaces of the 3 eyes were clear and smooth; the entire corneal surfaces were evenly covered with the transplanted cultivated corneal epithelium, which did not stain with fluorescein. The ocular surface epithelia of these patients are all stable without epithelial defects. CONCLUSIONS: We have shown that, in cases where the initially transplanted cultivated epithelium becomes opaque, it is possible to repeat the transplantation process with new cultivated epithelium on AM.     

新鲜羊膜移植在重建健康眼表面中的应用

目的：探讨新鲜羊膜移植在治疗严重的急、慢性眼表疾病中重建健康眼表的可行性，评价其疗效。方法：本院住院的眼表疾病患者21例（22只眼）分别进行单纯羊膜移植术13例（14只眼）、羊膜移植结膜囊成形术2例（2只眼）和羊膜移植联合自体角膜缘干细胞移植术6例（6只眼）。术后随访6-12个月，平均8.5个月。结果：所有移植的羊膜植片均未出现溶解和排斥，21例术后视力均提高。角膜溃疡患者未见角膜继续溶解，睑球粘连患者恢复眼球运动功能，翼状胬肉未见复发。结论：新鲜羊膜移植可有效应用于重建眼表面，有一定的临床推广价值。     

Relapsing Mooren's ulcer after amniotic membrane transplantation combined with conjunctival autografting

PURPOSE: To report a patient with Mooren's ulcer that relapsed 2 months after amniotic membrane (AM) transplantation and conjunctival autografting and its subsequent retreatment. DESIGN: Interventional case report. METHODS: We performed multilayered AM transplantation and conjunctival autografting from the opposite healthy eye to treat a corneoscleral perforation caused by Mooren's ulcer in a 60-year-old woman. MAIN OUTCOME MEASURES: Reformation of the anterior chamber, absence of inflammation, and restoration of visual acuity. RESULTS: The perforated corneoscleral lesion was sealed successfully by the AM and conjunctiva graft and led to a stable condition for 2 months. Relapsing corneal edema, keratic precipitates, and cell infiltration occurred along the margin of the conjunctival graft with severe vessel engorgement. After removing the conjunctival graft and regrafting of additional AM, the lesion subsided for at least 1 year. CONCLUSIONS: Amniotic membrane transplants may be useful in treating corneal perforation of immunologic origin, but conjunctiva and its vessels may play a role in the process of peripheral corneal destruction of Mooren's ulcer.     

羊膜培养液抑制角膜新生血管的实验研究

目的　研究羊膜培养液对小鼠角膜新生血管的抑制作用及机制。方法　应用cornealmicropocket方法 ,将含 10 0ng碱性成纤维细胞生长因子 (bFGF)和 12 %hydronpolymer的缓释颗粒植入角膜层间 ,制备小鼠角膜新生血管模型。实验分对照组、羊膜组和去上皮羊膜组 ,每组 10只眼 ,收集各组培养液于模型制作当日起 ,每日滴眼 4次至术后 7d摄片观测角膜新生血管长入情况并处死动物。对脐静脉血管内皮细胞 (HUVEC)进行体外培养 ,并应用Boyden小房技术和荧光结合(CyQUANT)实验分别检测羊膜培养液对HUVEC细胞迁移和细胞增殖的影响。利用酶联免疫吸附(ELISA)法检测各组培养液中金属蛋白酶抑制剂 1(TIMP1)和 2 (TIMP2 )的含量。结果　羊膜培养液明显抑制由bFGF诱发的小鼠角膜新生血管的形成 ,对照组、羊膜组和去上皮羊膜组角膜新生血管面积分别是 (2 4 8± 0 76 )mm2 、(0 6 4± 0 5 2 )mm2 和 (1 96± 0 6 5 )mm2 。羊膜培养液明显抑制血管内皮细胞的迁移和增殖 ,而对照组和去上皮羊膜组对HUVEC细胞迁移无作用。羊膜培养液中TIMP2水平明显增高 ,而TIMP1的含量无变化。结论　羊膜培养液中TIMP1的含量未增加。而羊膜上皮产生或释放大量的TIMP2 ,抑制血管内皮细胞的迁移和增殖 ,其可能是羊膜培养液抑制角膜新生血管的     

The successful culture and autologous transplantation of rabbit oral mucosal epithelial cells on amniotic membrane

PURPOSE: To determine the feasibility of using human amniotic membrane (AM) as a substrate for culturing oral epithelial cells and to investigate the possibility of using autologous cultivated oral epithelial cells in ocular surface reconstruction. METHODS: An ocular surface injury was created in one eye of each of eight adult albino rabbits by a lamellar keratectomy, and a conjunctival excision was performed, including and extending 5 mm outside the limbus. Oral mucosal biopsy specimens were obtained from these eight adult albino rabbits and cultivated for 3 weeks on a denuded AM carrier. The cultivated epithelium was examined by electron microscopy (EM) and immunohistochemically labeled for several keratins. At 3 to 4 weeks after the ocular surface injury, the conjunctivalized corneal surfaces of the eight rabbits were surgically reconstructed by transplanting the autologous cultivated oral epithelial cells on the AM carrier. RESULTS: The cultivated oral epithelial sheet had four to five layers of stratified, well-differentiated cells. EM revealed that the epithelial cells were very similar in appearance to those of normal corneal epithelium, had numerous desmosomal junctions, and were attached to a basement membrane with hemidesmosomes. Immunohistochemistry confirmed the presence of the keratin pair 4 and 13 and keratin-3 in the cultivated oral epithelial cells. Corneas that were grafted with the cultivated oral epithelial cells on an AM carrier were clear and were all epithelialized 10 days after surgery. CONCLUSIONS: Cultures of oral epithelial cells can be generated to confluence on AM expanded ex vivo from biopsy-derived oral mucosal tissue. Autologous transplantation was performed with these cultivated oral epithelial cells onto the ocular surfaces of keratectomized rabbit eyes. Autologous transplantation of cultivated oral epithelium is a feasible method for ocular surface reconstruction. The long-term outcome of such transplantation is not yet clear, and its feasibility in clinical use should be evaluated further.     

Transplantation of human limbal epithelium cultivated on amniotic membrane for the treatment of severe ocular surface disorders

PURPOSE: To study the short-term clinical results of transplanting of cultivated corneal/limbal epithelial cells on human amniotic membrane (AM) for limbal deficiency. DESIGN: Noncomparative, retrospective interventional case series. PARTICIPANTS: Thirteen eyes of 13 patients with severe limbal deficiency (Stevens-Johnson syndrome in eight eyes, ocular cicatricial pemphigoid in three eyes, and chemical burns in two eyes) were treated at the department of Ophthalmology, Tokyo Dental College, Japan. INTERVENTION: Cultivated allo-limbal epithelium was transplanted onto the ocular surface of patients with severe limbal deficiency. MAIN OUTCOME MEASURES: Ocular surface reconstruction with corneal epithelialization, changes in visual acuity, and postoperative complications were studied. Histologic examinations were also performed on cultivated epithelium. RESULTS: Cultivated corneal epithelium on AM formed two to three layers with the formation of basement membrane-like structures. After the surgery, the epithelium regenerated and covered the ocular surface in eight eyes (61.5%). However, three of the eight eyes developed partial conjunctival invasion, and two eyes later developed epithelial defects. At last examination, corneal epithelialization was achieved in six eyes (46.2%). Five eyes had conjunctivalization, one eye had dermal epithelialization, and one eye was not epithelialized. Complications were corneal perforation in four eyes and infectious keratitis in two eyes. CONCLUSIONS: This study demonstrates that the success rate for transplanting cultivated allo-limbal epithelium on the AM is not different from the conventional limbal and AM transplantation for the treatment of severe limbal stem cell dysfunction.