羊膜培养兔角膜缘上皮细胞及自体移植--角膜缘干细胞缺损的治疗

目的　探讨以人羊膜为载体培养兔角膜缘上皮细胞及其自体移植治疗全角膜缘干细胞缺损。方法　在8只兔右眼用正庚醇脱上皮和角膜缘环切的方法构建全角膜缘干细胞缺损模型2月。其中6只兔为实验组,活体取左眼角膜缘浅层小块,置羊膜上常规和气_液培养42天后进行自体移植治疗右眼角膜缘干细胞缺损;2只兔为对照组,直接用解冻无细胞人羊膜移植治疗右眼角膜缘干细胞缺损。进行细胞和术眼活体观察、组织学观察和电镜观察。结果　角膜缘上皮细胞在羊膜上生长良好,形成复层,细胞间的联结结构存在,细胞与羊膜组织粘附牢固。实验组移植手术后角膜迅速上皮化,恢复角膜表面光滑和透明,组织学观察和电镜观察呈现生理角膜上皮层的结构特点。但眼睑闭合不全可导致手术失败。对照组术后出现角膜缘干细胞缺损导致的角膜病变。结论　以羊膜为载体培养角膜缘上皮细胞后自体移植可有效地治疗角膜缘干细胞缺损导致的角膜病变。     

培养角膜缘干细胞羊膜移植治疗碱烧伤动物的实验研究

目的 观察培养生长于羊膜的角膜缘干细胞移植的治疗角膜缘碱烧伤伤的效果。方法 将兔角膜缘干细胞在的代培养后接种于羊膜,对新西兰大白兔角膜缘碱烧伤动物模型行角膜缘干细胞羊膜移植术,并对治疗后的角膜进行临床及病理学检查。结果 体外培养的兔角膜缘士细胞可在羊膜上继续增殖、分化为密集的角膜上皮细胞层;角膜缘干细胞移植术后兔角膜缘轻度充血、角膜上皮完整基质细胞浸润减轻、新生血管减少。组织病理学染色证实,角膜缘     

培养兔自体角膜缘干细胞移植的实验研究

目的 观察以羊膜为载体的兔角膜缘于细胞膜片移植治疗兔角膜缘于细胞缺损的效果。方法 制造兔角膜缘干细胞缺损的动物模型,以右眼为实验眼,从兔左眼取角膜缘组织,将兔角膜缘干细胞消化下来,接种于铺有羊膜的无菌六孔培养板中,待细胞形成多层角膜上皮细胞后,对兔角膜缘干细胞缺损动物模型行角膜缘干细胞羊膜移植术,并对治疗后的角膜进行裂隙灯及病理学检查。结果 临床和组织病理学染色证实：体外培养的兔角膜缘干细胞可在羊膜上继续增生、分化为多层角膜上皮细胞;角膜缘干细胞移植术后兔角膜缘轻度充血、角膜上皮完整、基质细胞浸润减轻、新生血管减少或消失。结论 应用角膜缘干细胞羊膜移植术可恢复其角膜上皮结构的完整性,减少角膜新生血管的形成,维持角膜缘的细胞屏障功能。     

不同移植手术治疗角膜缘干细胞缺损的实验研究

目的探讨以人羊膜为载体培养的角膜缘干细胞，自体及异体移植治疗全角膜缘干细胞缺损。方法制作兔眼角膜缘干细胞完全缺损3个月的模型。实验动物随机分为自体移植组和异体移植组，前者取对侧眼角膜缘组织，后者取异体兔眼角膜缘组织，均以去除上皮细胞的羊膜基底膜为载体，培养12d后行角膜缘干细胞羊膜移植术。术后观察3个月，以角膜上皮染色、角膜浑浊和新生血管3项指标进行临床疗效评定，通过病理检查评估术后角膜上皮修复情况，印迹细胞学检查移植前后角膜上皮的细胞表型。结果体外培养的兔角膜缘干细胞可在羊膜上粘附生长并增生，体外培养12d可形成复层。自体移植组和部分异体移植组术后角膜上皮逐渐愈合，透明度提高，基质细胞浸润减轻，新生血管减退或消失。印迹细胞学检查显示：移植前角膜上皮细胞PAS阳性，而移植后转为阴性；组织病理学显示：移植前角膜上皮大部分缺损，移植后呈现角膜上皮结构。部分异体移植组术后出现了免疫排斥反应。结论兔自体角膜缘干细胞羊膜移植术可重建眼表；免疫排斥反应仍是异体角膜缘干细胞羊膜移植术失败的主要原因。     

兔角膜缘上皮细胞培养后自体移植修复

目的：运用培养角膜缘上皮细胞联合人羊膜行自体移植的方法，观察植片修复兔眼角膜上皮的疗效。方法：选用健康新西兰白兔20只，制成右眼角膜缘干细胞缺乏的兔眼模型，其中12只兔行角膜缘上皮细胞培养联合羊膜自体移植，另外8只兔只进行单纯羊膜移植。术后每周对眼表情况进行评分，术后1mo眼角膜进行苏木素-伊红(HE)染色和透射电镜观察。结果：移植了含有自体角膜上皮细胞的兔眼，术后早期都形成了角膜上皮化并明显抑制了新生血管的再生，HE染色和电镜观察表明培养并移植的角膜上皮与正常的角膜上皮无明显差异；而只接受羊膜移植的兔眼，术后又出现角膜混浊和明显的新生血管，表明角膜表面被结膜上皮覆盖。结论：该方法术后早期可以恢复角膜上皮化，重建正常眼表，疗效明显优于单纯羊膜移植。     

兔同种异体角膜缘移植角膜印迹细胞学检测

目的 研究兔角膜缘干细胞缺乏和同种异体角膜缘移植后临床和角膜上皮表型的改变。方法 建立兔角膜缘干细胞缺乏模型,1个月后对治疗组进行同种异体角膜缘移植,术后联合使用免疫抑制剂。比较治疗组与非治疗组的临床表现和角膜表型的改变。结果 兔角膜缘干细胞缺乏后,角膜混浊,新生血管化,持续性上皮缺损;角膜上皮为结膜细胞表型。移植术后角膜上皮完整,新生血管减少,角膜透明度增加;上皮恢复角膜表型。结论 兔同种异体角膜缘移植联合术后使用免疫抑制剂是治疗角膜缘干细胞缺乏症的有效方法。印迹细胞学检查是角膜缘干细胞缺乏症诊断和角膜缘移植术后的评价手段。     

组织工程角膜上皮移植治疗兔角膜碱烧伤的形态学观察

目的:探讨组织工程角膜上皮移植治疗角膜碱烧伤的疗效和时机。方法:1mol/LNaOH制作改良兔角膜碱烧伤模型21只42眼,分对照组和移植组,移植组分别在碱烧伤后1,3,6,9d(早)和14d(中)行自体或同种异体组织工程角膜上皮移植术,比较移植组及对照组烧伤后28d内眼表和组织病理学变化。结果:角膜碱烧伤后7d开始出现角膜上皮的大片脱落,14d角膜上皮大片脱落或溃疡发生率达72%,持续至28d,而移植组在28d时发生率仅为25%,大多获得完整的角膜上皮;烧伤后早期移植组角膜基质深层炎性细胞浸润和新生血管生长较对照组明显受到抑制,而中期移植组角膜基质层较对照组并无明显差异;28d内异体组织工程角膜上皮移植的免疫排斥反应并不大于自体移植。结论:自体或同种异体组织工程角膜上皮移植均可尽快恢复眼表完整性,且烧伤后早期移植效果明显优于中期移植。     

深低温保存角膜缘干细胞自体移植实验研究

目的:评价深低温冷冻保存兔眼角膜缘干细胞活性及自体移植治疗角膜干细胞缺乏眼表疾病的疗效。方法:12只新西兰白兔用刀片制备带2mm周边角膜和2mm球结膜的环行浅层角膜缘植片,去除余下的角膜上皮,制成角膜干细胞缺乏眼表疾病模型。角膜缘植片通过程序降温仪程序降温后-196℃深低温保存。30天和60天后作自体移植行兔眼眼表重建术。结果:角膜干细胞缺乏导致角膜上皮愈合延迟,7眼在术后22～26天愈合,平均24±1天,4眼复发性上皮糜烂;基质混浊水肿;角膜血管化,平均5±1天出现新生血管。深低温保存角膜缘干细胞自体移植能促进角膜上皮愈合,平均10±2天愈合,基质混浊逐渐吸收,新生血管消退、变细,2眼消失。结论:深低温保存法能保存角膜缘干细胞活性;能随时按需为临床提供活性角膜缘材料;为临床深低温保存角膜缘干细胞移植治疗眼表疾病提供了实验依据。眼科学报1998;14:224～226。     

不同诱导剂对体外角膜缘干细胞培养的影响

眼表重建常用的羊膜移植或自体角膜缘干细胞移植的方法,对眼表组织损伤过大,且对眼表自身干细胞缺乏者临床效果较差。我们常采用体外培养自体角膜缘干细胞后联合生物载体进行眼表移植重建。本实验对兔角膜缘干细胞进行体外培养,观察不同的诱导环境对于细胞最终的繁殖分化的影响。     

组织工程角膜上皮治疗兔角膜缘干细胞缺乏症的研究

目的 评价以纤维凝胶膜为载体,体外培养自体角膜缘干细胞移植术治疗角膜缘干细胞缺乏症的临床效果。方法 制作兔眼角膜缘干细胞完全缺乏模型,取对侧眼角膜缘组织进行干细胞培养,并以纤维凝胶膜为载体制成组织工程角膜上皮。将实验动物随机分为4组,其中Ⅰ-Ⅲ组为实验组,行组织工程角膜上皮移植术,Ⅰ组移植术后观察3个月,Ⅱ组移植术后观察1个月,Ⅲ组移植术后观察2周。Ⅳ组为对照组,移植不含干细胞的凝胶膜,术后观察3个月。以角膜上皮染色、角膜混浊及新生血管3项指标进行临床疗效评定,通过病理检查评估术后不同时期角膜上皮修复情况,印迹细胞学检查移植前后角膜上皮的细胞表型,免疫组织化学染色观察角膜上皮特异性角蛋白K3、MUCSAC及转录因子p63在移植角膜上皮的表达。结果 实验组角膜上皮逐渐愈合,透明度提高,新生血管减退或消失;对照组角膜呈持续混浊,上皮缺损迁延不愈,最终大量新生血管长入,上皮结膜化。临床指标评分比较,差异有统计学意义（P=0．021）。角膜上皮印迹细胞学检查显示对照组为PAS（＋）的结膜细胞表型,而实验组上皮细胞PAS（-）。组织病理学显示实验组为正常角膜上皮结构,对照组为结膜化生的上皮,可见血管和杯状细胞。免疫组织化学显示实验组角膜上皮表达角膜上皮特异性角蛋白l（3（AE5）,不表达结膜特异性MUCSAC,在上皮基底部表达转录因子p63。对照组上皮持续表达MUCSAC。结论 组织工程角膜上皮移植术是一种有效的治疗角膜缘干细胞缺乏症的方法,可重建眼表,修复角膜上皮的损伤,抑制角膜上皮结膜化和新生血管。纤维凝胶膜为一种新型的组织工程材料,吸收快,透明度高,具有良好的组织相容性,是一种较理想的移植载体材料。（中华眼科杂志．2006．42：679-685）     

带角膜缘板层角膜移植治疗眼表烧伤

目的探讨带角膜缘板层角膜移植治疗[表角膜烧伤及疗效。方法采用带角膜缘板层角膜移植治疗化学伤、热灼伤56例58[,其中[睑皮肤缺损32例32[、睑球粘连36例38[;术前角膜印迹细胞学检查,角膜表面有杯状细胞56例58[。结果术后视力提高46[,角膜上皮保持完整48[,因[睑缺损而影响角膜上皮愈合,行睑裂闭合手术10[,术后排斥21[,用Cs-A或FK-506、皮质激素控制,对术后发生青光[中的6[进行手术,术后板层角膜表面行印迹细胞检查出现杯状细胞19[。结论带角膜缘板层角膜移植,可用于严重[表烧伤的角膜结膜化病人。酸性化学伤、[附属器完好、[表植床条件好的,手术后角膜上皮覆盖完好,角膜再度结膜化程度低。     

翼状胬肉手术中自体角膜缘干细胞移植与生物羊膜移植的对比

目的观察翼状胬肉手术中自体角膜缘干细胞移植与生物羊膜移植的效果。方法72例（84眼）翼状胬肉随机分为自体角膜缘干细胞移植组（A组）和生物羊膜移植组（B组）。术中首先进行翼状胬肉切除,之后分别进行自体角膜缘干细胞移植或生物羊膜移植。随访6～24个月。结果A组术后角膜上皮平均于（3．19±0．65）d愈合,B组术后角膜上皮平均于（6．22±1．35）d愈合,（P〈0．05）。A组术后1眼复发,复发率为2．38％,B组术后3眼复发,复发率为7．14％（P〉0．05）。结论自体角膜缘干细胞移植与生物羊膜移植均能降低翼状胬肉术后复发率,其中自体角膜缘干细胞移植更易于角膜创面愈合。     

Limbal stem cell transplantation in chronic inflammatory eye disease

OBJECTIVE: The goal of this study was to describe the outcome of limbal stem cell transplantation (LSCT) in patients with severe ocular surface disease caused by underlying chronic inflammatory eye disease. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: Nine patients with limbal stem cell deficiency caused by an underlying ocular inflammatory disease who underwent LSCT. METHODS: The authors reviewed the records of 11 eyes of 9 patients with immunologically mediated ocular surface disease that underwent LSCT. MAIN OUTCOME MEASURES: The main outcome measures were reepithelialization of the corneal surface, restoration of corneal surface, and improvement in visual acuity. RESULTS: A total of 11 eyes underwent either autologous (n = 1) or HLA-matched living related donor (n = 10) LSCT for ocular surface disease secondary to inflammatory disease. Reepithelialization of the corneal surface in the immediate postoperative period occurred in 10 eyes (91%) within an average of 10 days (range, 3-21 days). Long-term restoration of the corneal surface was achieved in six (55%) eyes. Visual acuity improved in six eyes (55%). Reasons for poor outcomes included microbial infection, limbal stem cell graft rejection, and corneal ulceration. No donor eyes had complications. CONCLUSIONS: Patients with underlying immunologically mediated diseases, such as Stevens-Johnson syndrome, toxic epidermal necrolysis, or ocular cicatricial pemphigoid, who undergo LSCT have lower success rates than do those patients with noninflammatory ocular surface diseases.     

复发性翼状胬肉扩大切除及干细胞移植的效果

目的评价复发性翼状胬肉扩大切除联合自体角膜缘干细胞移植及丝裂霉素C术的临床效果。方法回顾性分析42例（46眼）复发性翼状胬肉,实施扩大切除、自体角膜缘干细胞移植联合丝裂霉素C应用,术后随访6～24月,观察翼状胬肉复发率及眼表情况。结果联合手术后2眼有复发倾向,占4．35％。眼表术后反应轻。结论翼状胬肉扩大切除联合自体角膜缘干细胞移植及丝裂霉素C应用治疗复发性翼状胬肉能够有效降低胬肉的复发率。     

角膜缘上皮移植治疗化学伤和热烧伤后的眼球表面疾患

本文采用自体或异体角膜缘上皮移植治疗８例（８眼）化学伤和热烧伤后角膜表面缺损及血管翳性混浊。结果：８例中，上皮稳定愈合７例，角膜透明６例（其中包括１例行Ⅱ期穿透性角膜移植术），视力均有明显提高。结论：采用自体或异体角膜缘上皮移植替换严重受损的角膜缘组织，通过供体干细胞的增殖、分化能有效地修复和稳定受损的角膜表面，是治疗单侧或双侧眼球表面疾患的合理治疗方法     

Amniotic membrane graft in ocular surface disease. Prospective study with 31 cases

INTRODUCTION: Amniotic membrane's unique combination of properties including the facilitation of migration of epithelial cells, the reinforcement of basal cellular adhesion and the encouragement of epithelial differentiation [6] together with its ability to modulate stromal scarring and its anti-inflammatory and anti-bacterial activity has led to its use in the treatment of ocular surface pathology as well as an adjunct to stem cell grafts of the corneal limbus [6-4]. We report a prospective study of 30 patients so treated. MATERIAL AND METHODS: We studied 31 eyes of 30 patients subjected to amniotic membrane grafts between September 1999 and May 2000. There were 25 men and 5 women with an average age of 60.1 (range 25-86) years who were followed for a mean of 7.7 (range 4-11) months. 5 groups (A to D) were observed: A: 6 eyes. Small chronic ulcers without limbal involvement. B: 4 eyes. Ulcers of at least 75% corneal area or occupying 75% of the limbus. C: 9 eyes. Corneal burns. D: 8 eyes. Painful bullous corneal dystrophies unresponsive to other treatment. E: 4 eyes. Symblepharons. Amniotic membrane was placed on the corneal lesion, epithelial surface externally [6, 15], trimmed and sutured with interrupted 10/0 nylon, removed at one month. In two patients (11, 12) inflamed conjunctiva was recessed and amnion sutured to the recessed margin. For the bullous dystrophies we removed all the corneal epithelium and either sutured the amnion to peri-limbal conjunctiva (4 eyes) or to the limbus (4 eyes). For the symblepharons the conjunctiva was dissected to reform the fornix which was lined with amniotic membrane, sutured with 8/0 vicryl. Patients were reviewed regularity. RESULTS: Group A: All healed within 15 days, in most with dissolution of the amnion over 2-3 months although some persisted, covered with corneal epithelium. An eye with a Descemetocoele and one with a microperforation both healed. Vision improved more than two lines in 4 of 6 eyes. Group B: 2 of 4 eyes healed, one despite detachment of the membrane after 15 days. One eye was salvaged by tarsorrhaphy over a fresh keratoplasty after perforation of a neuroparalytic ulcer on failure of three successive amnion grafts. The final cornea vascularised despite an amnion graft for a meta-herpetic ulcer. Group C: 2 of 9 eyes had limbal damage in one quadrant but 7 had vessels in at least three-quarters of the circumference. One (15) also had a limbal autograft. 3 of 9 eyes healed satisfactorily with more than 2/10 improvement in acuity in each case. 2 showed further neovascularisation despite surface healing. One old chemical burn healed satisfactorily but vascularisation remained 5 eyes failed to heal with lysis of the graft, the patient who had a limbal autograft developed a vascular pannus, and in 4 eyes neovascularisation progressed to cover the entire cornea. Group D: 3 eyes settled with loss of symptoms but in 5 the graft detached within 15 days. All eyes where the membrane had been sutured to the conjunctiva beyond the limbus failed whilst 3 of 4 in which it had been sutured anterior to the limbus succeeded, leaving a persistent whitish membrane under the epithelium. Group E: We were able to reconstruct the cul de sac in 3 out of 4 eyes. In one patient with recurrent pterygium good ocular movement was restored, previously limited by scarring. One with associated ocular surface damage from a thermal burn failed by scarring of the cul de sac a month after surgery. DISCUSSION: Our best results were in persistent trophic ulcers of the cornea (Groups A and B) with a success rate of 80%, comparable to those of others [49, 37, 38]. The ready availability of amniotic membrane in our facility makes amniotic membrane transplantation the main secondary treatment for such lesions, especially because of the visual improvement we obtained. Because we did not observe any improvement in corneal thickness after this treatment we advise its early use before significant stromal lysis. The technique was not sufficient to control the effect of corneal anaesthesia in two eyes [40] or in chemical burns suggesting that amniotic membrane alone is insufficient to promote corneal healing in the absence of limbal stem cells. Nevertheless, three eyes did benefit. It has been suggested [13] that the anti-apoptotic function of amnion may prevent stem cell loss in such eyes [42], thus it appears logical to offer an amniotic membrane graft first, before stem cell transplantation, which may entrain complications in the donor eye if autografted [43] or because of the rejection risk of an allograft. It may be that an amniotic membrane graft simply becomes a holding procedure allowing time to settle the eye so as to allow secondary procedures to address the underlying cause of further damage. Our treatment of bullous dystrophy only succeeded on confining the graft to within the limbus, 3 out of 4 eyes becoming comfortable. By contrast we found amniotic membrane helpful in reconstructing symblepharons in the absence of local inflammation. CONCLUSION: Amniotic membrane grafting is a simple and straightforward surgical technique which should form part of the therapeutic arsenal for the treatment of ocular surface disease. Indications for the technique need further clarification for it is evident that it cannot correct all secondary pathology associated with limbal destruction. It is certainly preferable to conjunctival advancement and has proved useful in the reconstruction of the cul-de-sac.     

Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease

PURPOSE: To bioengineer a corneal surface replacement using ex vivo expanded, cultured corneal epithelial stem cells seeded on a matrix derived from amniotic membrane and use this bioengineered graft to manage difficult ocular surface disease. METHODS: Fourteen patients with ocular surface disease unresponsive to standard medical and surgical treatments, including seven patients with presumed limbal stem cell deficiency were chosen for transplantation of a bioengineered composite corneal surface in eye each. Presumed corneal stem cells were harvested from either the patient's or related donor's limbus, expanded ex vivo, and cultivated on a carrier of modified human amniotic membrane. The resulting composite cultured tissue was transplanted to the ocular surface of the diseased eye, from which the abnormal tissue had been surgically removed. Ten patients received autologous grafts, and four received allogeneic grafts. RESULTS: A successful outcome, defined as restoration or improvement of vision, along with maintenance of corneal re-epithelialization and absence or recurrence of surface disease was obtained in 6 of the 10 patients with autologous procedures and in all 4 allogeneic transplants. Follow-up ranged 6-19 months with a mean of 13 months. CONCLUSIONS: This novel technique documents that presumed corneal epithelial stem cells can be harvested safely from the limbus, expanded successfully in vitro, and grown on denuded amniotic membrane. The resultant composite cultured tissue can be transplanted and appears to successfully manage eyes with difficult ocular surface disease, including those with stem cell deficiency. This technique minimizes the threat of damage or depletion to the contralateral or donor limbus.     

Limbus transplantation for reconstruction of the ocular surface

Proliferation of the corneal epithelium originates in undifferentiated, long-lived stem cells that are located in the basal limbal epithelium. Stem cells are important for corneal epithelial regeneration and wound healing. Depletion of stem cells due to accidents as well as malfunctions of stem cells due to inborn or inflammatory diseases result in limbal stem cell deficiency. Limbal deficiency is characterized by conjunctivalization of the cornea with vascularization and opacification. Partial limbal deficiency can be treated by removing ingrown conjunctival epithelium thus allowing normal limbal epithelium to repopulate the cornea. Unilateral limbus-derived stem cell disease requires either limbal autograft transplantation from the healthy partner eye or kerato-limbal allograft transplantation. Several modifications of the latter technique have been performed including large kerato-limbal lamellar grafts and central penetrating kerato-limbal allografts. All homologous procedures render a very high risk of immunological reactions that require long term systemic immunosuppression. The use of amniotic membrane, better pharmacological drugs for immunosuppression and improvements in the HLA-matching of limbal allografts as well as ex vivo expansion of corneal stem cells should allow for better reconstruction of the ocular surface in limbal deficiency.