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。     

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。     

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。     

异体角膜缘上皮细胞培养后移植的实验研究

目的观察含培养的角膜缘上皮细胞的羊膜片移植到角膜缘干细胞损伤的异体兔眼角膜上的效果。方法把羊膜为载体组织块培养的角膜缘上皮细胞移植到角膜缘干细胞损伤的异体兔眼角膜上,术后观察角膜混浊度、角膜新生血管、角膜上皮等情况,定期兔进行病理组织学检查。结果角膜缘上皮细胞在羊膜上培养16d形成3～4层,用含有培养的异体角膜上皮细胞的羊膜片移植的12只兔眼,术后第5天,损伤的角膜表面全部上皮化,第16天开始,12只兔眼逐渐出现排斥反应。结论含培养的角膜缘上皮细胞羊膜片异体移植术后早期角膜全部上皮化,晚期则出现排斥反应。     

壳聚糖复合共混膜培养兔角膜缘上皮及其移植

目的 探讨壳聚糖、胶原、透明质酸钠复合共混膜作为兔角膜缘上皮细胞体外培养载体及其自体移植的可行性。方法 活体取兔左眼角膜缘浅层小块，置此共混膜上，常规培养8天。将兔右眼角膜浅层基质分离成囊袋，用3mm环钻去除角膜中央浅表层，将含培养角膜缘上皮细胞的共混膜植入自体角膜囊袋内(6只兔)，对照组则植入不含细胞的共混膜(6只兔)。术后观察1月。结果 角膜缘小块在共混膜上培养生长良好，移植手术后40h，实验组5／6角膜植片荧光素染色阴性；对照组术后4天角膜植片荧光素染色仍为部分阳性。实验组组织学检查显示材料表层上皮完整，AE1／AE3免疫组化染色阳性，材料部分降解。结论 壳聚糖胶原透明质酸钠共混膜可作为培养角膜缘上皮细胞及其移植的载体。     

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     

外伤性角膜缘干细胞缺陷症手术治疗的实验研究

目的 探讨兔不同类型的外伤性角膜缘干细胞缺陷的手术治疗方法及临床疗效。方法 建立兔完全性角膜缘干细胞缺陷和部分性角膜缘干细胞缺陷模型，7天后分别给予羊膜移植、自体角膜缘移植和羊膜 自体角膜缘移植，比较各组治疗后的临床表现。结果 兔完全性角膜缘干细胞缺陷组羊膜移植失败，而自体角膜缘移植和羊膜 自体角膜缘移植恢复了角膜上皮完整性和角膜缘屏障功能；部分性角膜缘干细胞缺陷组羊膜移植、自体角膜缘移植和羊膜 自体角膜缘移植均达到了重建眼表的目的。结论 对于角膜缘缺陷患者，必须了解其角膜缘干细胞缺乏程度，然后分别给予不同的治疗。     

人骨髓间充质干细胞在兔角膜缘干细胞缺损动物模型中的分化

目的探讨人骨髓间充质干细胞（mesenchymal stem cells,MSCs）分化为角膜上皮细胞的可塑性及可行性。方法20只日本大耳白兔随机分为对照组和实验组,每组10只,右眼为实验眼。实验组：将人MSCs接种在人羊膜上培养4d后移植到兔角膜缘干细胞缺损的动物模型;对照组：仅采用羊膜移植到兔角膜缘干细胞缺损的动物模型。分别于移植后第1、第2、第3、第4和第6周,摘取各组眼球,分别行HE和PAS染色、单克隆抗体AE1和PCNA染色、透射电镜和扫描电镜检查。结果人MSCs接种到羊膜后能在羊膜上生长,与羊膜共培养4d后,人MSCs贴附羊膜生长迅速,组织学特征无明显改变。羊膜负载人MSCs移植到兔角膜基质表面,角膜表层细胞PAS染色呈阴性,AE1和PCNA染色呈阳性,具有典型的上皮细胞形态;而对照组PAS染色呈阳性,AE1和PCNA染色呈阴性。超微结构观察可见,实验组电镜下可见微绒毛、桥粒和张力丝等典型上皮细胞结构,而对照组未见以上明显特征。结论羊膜负载人MSCs移植到兔眼表角膜基质后,MSCs能存活、增殖并向角膜上皮样细胞分化。     

自体角膜缘干细胞联合羊膜移植治疗眼表疾病

目的:探讨角膜缘干细胞联合羊膜移植治疗眼表疾病的临床效果。方法:对翼状胬肉420例(452眼),持续性角膜上皮缺损合并基底部溃疡20例(20眼),采用自体角膜缘干细胞移植联合人羊膜移植术进行治疗,并随访观察1～7a。结果:翼状胬肉452眼,上皮愈合良好,角膜病变区光滑透明,18眼复发,复发率为4.0%。持续性角膜上皮缺损合并基底部溃疡21眼,角膜溃疡修复、上皮缺损区界面光滑平整、清亮。结论:角膜缘干细胞为病灶角膜提供了健康干细胞来源,恢复了角结膜屏障功能;人羊膜本身无抗原性,术后不发生免疫排斥反应,且具有抗炎、抑制新生血管及抗纤维化作用;在促进伤口愈合中起重要作用,使其成为角膜上皮移行及生长的支架。自体角膜缘干细胞联合羊膜移植是一种有效的治疗眼表疾病的手段。相对于角膜缘干细胞而言,羊膜仅起着角膜上皮生长移行支架物的作用,角膜缘干细胞移植是不能被羊膜移植所替代的。     

组织工程口腔黏膜上皮治疗角膜缘干细胞缺陷症的实验研究

目的探讨体外培养的自体组织工程口腔黏膜上皮重建兔角膜上皮的可行性。方法制作兔角膜缘干细胞缺陷模型32只,实验组Ⅰ～Ⅲ以自体口腔黏膜上皮细胞为种子细胞制作组织工程上皮,移植到实验组模型兔角膜表面,分别观察2周、1个月、3个月,对照组移植空白载体膜,观察3个月。术后裂隙灯显微镜下观察,以角膜新生血管、混浊度及上皮染色评分评价移植效果。用组织病理、免疫组织化学和印迹细胞技术评价角膜上皮重建的可能性。结果模型兔角膜混浊,有大量新生血管和杯状细胞。实验组移植后角膜透明,印迹细胞检查PAS(-)。实验组与对照组术后角膜总评分差异有统计学意义(P=0.000),p63表达阳性,角膜上皮的组织特点及角蛋白的表达与正常角膜上皮相似。结论组织工程口腔黏膜上皮在角膜基质微环境的诱导下可分化为角膜样上皮细胞,有重建角膜上皮的作用。     

Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits

PURPOSE: To examine the viability of using human amniotic membrane as substrate for culturing corneal epithelial cells and transplanting them onto severely injured rabbit eyes. METHODS: An ocular-surface injury was created in the right eye of eight rabbits by a lamellar keratectomy extending 5 mm outside the limbus. Next, from the limbal region of the uninjured left eyes of five of these animals, a small biopsy of corneal epithelial cells was taken and cultured on acellular human amniotic membrane. One month later, the invading conjunctiva that covered the corneal surface of all eight injured eyes was surgically removed. Five of the eyes then received grafts of amniotic membrane containing autologous cultured epithelial cells, whereas the other three received grafts of acellular amniotic membrane alone. RESULTS: A confluent primary culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 14 days. Cells were partially stratified and fairly well attached to the underlying amniotic membrane, although a fully formed basement membrane was not evident. The three rabbits that received amniotic membrane transplantation alone all had total epithelial defects on the graft in the early postoperative period. Eyes that were grafted with amniotic membrane that contained cultivated epithelial cells, however, were all successfully epithelialized up to 5 days after surgery. CONCLUSION: Autologous transplantation of cultivated corneal epithelium is feasible by using acellular amniotic membrane as a carrier.     

羊膜移植与自体角膜缘干细胞移植治疗原发性翼状胬肉的临床疗效比较

目的羊膜移植与自体角膜缘干细胞移植治疗原发性翼状胬肉的临床疗效比较。方法将114例（131眼）随机分成A和B两组。A组53例（59眼）行翼状胬肉切除＋羊膜移植术,B组61例（72眼）行翼状胬肉切除＋自体角膜缘干细胞移植术,术后随访6-24个月,观察两组疗效并进行回顾性分析。结果 A组10眼复发,复发率为16.95%,B组6眼复发,复发率为8.33%,两组比较差异具有统计学意义（P〈0.05）,A组复发率明显高于B组。结论翼状胬肉切除＋自体角膜缘干细胞移植术,此手术取材方便、成功率高;而翼状胬肉切除＋羊膜移植术,手术耗材、成功率明显较自体角膜缘干细胞移植术低。     

Clinical outcome of autologous cultivated limbal epithelium transplantation

PURPOSE: To report the clinical outcome of autologous cultivated limbal epithelial transplantation. METHODS: Eighty-six patients' records and their clinical photographs were reviewed for demographics, primary etiology, type of limbal transplantation, ocular surface stability, visual acuity, final outcome and possible factors affecting outcome and complications. RESULTS: Eighty-eight eyes of 86 patients with limbal stem cell deficiency (LSCD) underwent autologous cultivated limbal epithelium transplantation between March 2001 and May 2003, with a mean follow-up of 18.3 months. The etiology of LSCD was alkali burns in 64% patients. Sixty-one eyes had total LSCD. Thirty-two of the 88 eyes had undergone amniotic membrane transplantation and 10 eyes had previously undergone limbal transplantation with unfavorable outcome. Nineteen eyes underwent penetrating keratoplasty, of which 11 grafts survived at the final follow-up. Finally, 57 eyes (73.1%, 95% CI: 63.3-82.9) had a successful outcome with a stable ocular surface without conjunctivalization, 21 eyes (26.9%, 95%CI: 17.1-36.7) were considered failures and 10 patients were lost to follow-up. CONCLUSION: LSCD can be successfully treated by autologous cultivated limbal epithelium transplantation in majority of the cases.     

Corneal surface reconstruction using adult mesenchymal stem cells in experimental limbal stem cell deficiency in rabbits

Purpose: To investigate the ability of mesenchymal stem cells (MSC) to transdifferentiate to corneal epithelial cells in experimental limbal stem cell deficiency in rabbits. Methods: Total limbal stem cell deficiency was produced in 21 right eyes of 21 New Zealand rabbits; 6 eyes served as controls (group 1, G1). After removal of the conjunctival overgrowth, five eyes received amniotic membrane transplantation (AMT; G2). In four eyes, autologous limbal stem cell transplantation from the healthy eye was performed with AMT (G3). In another six eyes, enriched autologous MSC were injected under the amniotic membrane (AM) (G4). Within 280 days, corneoscleral discs were analysed for goblet cells, cytokeratin (CK) 3/12, connexin 43, β₁‐integrin, CK 19, α‐enolase, p63 and ATP‐binding cassette transporter subtype G‐2 (ABCG‐2) distribution patterns. Results: Cultivated MSC were positive for CK 3/12 and α‐enolase, but negative for ABCG‐2, p63 and connexin 43. On rabbit corneas, CK 3/12 was expressed in all corneal regions in all groups, but with significantly different intensities. Among all other parameters, expression levels of ABCG‐2, β₁‐integrin and connexin 43 were significantly different between the transplanted groups and the control group. After a mean follow‐up time of 172 (47–280) days, goblet cells were rarely present in the central cornea (G1‐4). Conclusion: CK 3/12 is not highly specific for differentiated corneal epithelium. Further, goblet cells are not a reliable marker for conjunctivalization in rabbits. Expression of ABCG‐2, β₁‐integrin and connexin 43 after mesenchymal stem cell transplantation may indicate their ability to maintain their stem cell character or to transdifferentiate to epithelial progenitor cells.     

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。     

活体异体结膜缘和羊膜移植治疗化学性眼外伤造成的角膜缘干细胞缺失(英文)

目的:评价利用活体异体结膜缘和羊膜移植治疗化学性眼外伤造成的角膜缘干细胞缺失的临床效果。方法:从2005-07/2007-12,本研究包括了9名男性化学性眼外伤患者(10眼)。所有患者接受了亲属活体异体结膜缘和羊膜移植,2例眼接受了睑缝术。用环孢菌素和泼尼松龙进行全身性免疫抑制。结果:在3例眼中观察到完全角膜上皮化(30%),其中1例在术后1.5mo出现免疫排斥,角膜溶解引起穿孔,加大全身性免疫抑制剂量来控制病情。3例眼中植片无法在角膜表面重新形成上皮,被定为原发性失败。其余4眼有部分上皮形成,但上皮细胞无法完全覆盖角膜表面。术前最佳矫正视力从手动到1m处数指,术后最佳矫正视力从光感到20/80。有5眼视力得到改进,不需其他治疗。手术失败的主要原因为干眼症和持续性炎症。结论:对于能控制泪量和眼部炎症的病例,亲属活体异体角膜缘和羊膜移植是治疗化学性眼外伤造成的角膜缘干细胞缺失最佳方法之一。     

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.     

In vivo survival and stratification of cultured limbal epithelium

A 6-year-old Bangladeshi girl presented with total limbal stem cell deficiency in the left eye, secondary to a 6-month-old chemical injury. The patient had also previously undergone two limbal transplantation surgeries. At the authors' centre the child underwent autologous cultured limbal epithelium transplantation, on human amniotic membrane, without the use of air-lift technique. Symptomatic relief, re-epithelialization of the ocular surface, regression of corneal pannus and slight improvement in vision were all noted. The corneal button obtained at the time of keratoplasty (performed 4 months later) revealed stratified epithelium with basement membrane. Thirty-seven months post keratoplasty, the best-corrected visual acuity was 6/15 with clear graft and stable ocular surface. Herein, a case of limbal stem cell deficiency successfully managed by monolayer of cultured limbal epithelium is presented.