异种角膜脱细胞基质为供体进行角膜前板层移植的初步研究

目的探讨以异种猪角膜脱细胞基质为供体植片,分析兔角膜进行前板层移植后的生物相容性：设计实验研究。研究对象新西兰白兔。方法应用1％TritonX-100及冷冻干燥处理制备猪角膜脱细胞基质载体,切取1／3厚度前板层作为供体角膜植片,对兔眼角膜前板层切除后进行移植,同时以新鲜猪角膜板层为供体对兔进行前板层移植作对照。通过术后角膜透明度、组织结构观察,评价猪角膜脱细胞基质的生物相容性及植片转归的情况。主要指标角膜透明度和组织学HE染色。结果制备的猪角膜脱细胞基质植片作前板层移植到兔眼后,未见明显的新生血管、炎症反应、角膜坏死等排斥现象,观察期内植片较透明;脱细胞基质表面上皮化良好,植片基质板层与植床逐渐融合,植片内有宿主细胞迁入生长,板层结构与正常角膜相似。结论猪角膜脱细胞基质具有良好的生物相容性、安全性和低抗原性,有望成为角膜板层移植的供体材料。     

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.     

不同受者同期分别接受同者的角膜移植五例

目的 为提高角膜材料的利用率,探讨将同一噶供者的二个角膜同期移植给不同受者的可行性及手术方法.方法 供者的其中一个角膜材料分别为1例角结膜恶性黑色素瘤,1例蚕蚀性角膜溃疡伴穿孔行前部分板层角膜移植术,并对1例大泡性角膜病变行深板层角膜内皮移植术.另一个角膜材料分别为眼烧伤后重度睑球粘连行全板层角膜移植和1例大泡性角膜病变行深板层角膜内皮移植术.结果 5例角膜病患者手术均获得成功,其中2例大泡性角膜病变患者术后1个月,角膜上皮水泡消失,角膜水肿减轻,内皮植片透明.1例角结膜恶性黑色素瘤患者,视力术前0.3提高至术后0.6,植片透明,角膜植床及结膜无色素残留.1例蚕蚀性角膜伴穿孔经羊膜移植联合部分板层角膜移植治愈.1例眼烧伤睑球粘连患者,术后1个月,睑球粘连完全解除,植片透明,视力由术前0.1提高到0.3.结论 将同一供者的角膜分别移植给不同受者,能充分利用供者角膜材料,方法可行,效果良好.     

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

AIM: To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts, and an acellular porcine cornea matrix (APCM) in vitro. METHODS: The scaffold was prepared from fresh porcine corneas which were treated with 0.5% sodium dodecyl sulfate (SDS) solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin (HE) staining and 4’, 6-diamidino-2-phenylindole (DAPI) staining. Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM, and then cell proliferative ability was evaluated by MTT assay. To construct a human corneal anterior lamellar replacement, corneal fibroblasts were injected into the APCM and cultured for 3d, followed by culturing corneal epithelial cells on the stroma construction surface for another 10d. The corneal replacement was analyzed by HE staining, and immunofluorescence staining. RESULTS: Histological examination indicated that there were no cells in the APCM by HE staining, and DAPI staining did not detect any residual DNA. The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells. At 10d, a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed, and the injected corneal fibroblasts distributed within the scaffold. The phenotype of the construction was similar to normal human corneas, with high expression of cytokeratin 12 in the epithelial cell layer and high expression of vimentin in the stroma. CONCLUSION: Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix. This laid the foundation for the further transplantation in vivo.     

种植人骨髓间充质干细胞的猪角膜板层移植治疗兔角膜损伤的初步研究

目的研究种植人骨髓间充质干细胞（MSCs）的猪角膜基质治疗兔角膜损伤的可能性。方法用全骨髓贴壁法分离纯化人MSCs并传代,流式细胞仪检测免疫表型及诱导成脂、成骨分化鉴定。12只新西兰白兔随机分为2组,实验组取第3代MSCs接种于去上皮的猪角膜基质上,培养4 d后移植到广泛损伤的兔角膜上,对照组单纯移植去上皮猪角膜基质。术后2、4、8周,取各实验眼行组织学检查,观察移植的MSCs及猪角膜基质的存活、转归及移植局部的反应。免疫组织化学、免疫荧光染色检测移植后角膜上皮细胞角蛋白12的表达。结果培养获得的MSCs中CD29阳性者占95.97%,CD44阳性者占96.49%,CD90阳性者占92.79%,CD105阳性者占94.66%,CD34阳性者占0.59%,CD45阳性者占0.36%,符合MCSs的免疫表型,并可以诱导成脂及成骨分化。实验组MSCs接种到去上皮猪角膜基质后贴附、生长迅速,术后植片在植床上存活良好,无排斥反应,角膜较对照组透明,新生血管少,而对照组在移植后发生排斥反应。实验组角膜免疫组织化学及免疫荧光染色均检测出CK12阳性细胞。结论种植MSCs的猪角膜基质移植到损伤兔角膜后可以存活,MSCs可以分化为角膜上皮样细胞,具有构建组织工程角膜的潜能。     

组织工程角膜基质的体外构建及移植的实验研究

目的探讨利用组织工程技术体外构建角膜基质进行板层角膜移植的可行性和有效性。方法将猪角膜基质去细胞处理后制备成组织工程角膜基质载体;取幼兔角膜基质细胞体外培养,将其种植在载体上,体外构建成组织工程角膜基质,用PKH26荧光标记兔角膜基质细胞示踪角膜基质的构建;将16只兔的角膜基质内植入壳聚糖膜使之形成无菌性角膜溃疡,随机从16只兔中选8只,进行组织工程角膜基质移植;另外8只作为对照组,进行新鲜的同种异体兔板层角膜移植。术后对角膜进行裂隙灯、光学显微镜、透射电镜观察。结果体外构建的组织工程角膜的基质细胞具有活性,其结构与正常角膜基质相近。移植治疗无菌性角膜溃疡术后,1~2周有新生血管侵入组织工程角膜基质植片边缘,植片为灰白色半透明状;3~4周随着新生血管减退,组织工程角膜基质植片局部开始透明变薄;术后8~10周角膜溃疡完全修复,角膜恢复透明性,角膜神经可再生;观察最长达10月,角膜仍保持透明,无免疫排斥发生,与对照组疗效相同。结论体外构建的组织工程角膜基质无免疫原性、具有良好的生物相容性,可作为临床治疗角膜溃疡的移植材料。     

复方倍他米松对异种角膜板层移植术后免疫排斥反应的影响

目的：观察结膜下注射复方倍他米松对鸵鸟-兔眼板层角膜移植术后免疫排斥反应的缓释治疗作用。

方法：16只6周龄健康新西兰白兔角膜(单眼)行板层角膜移植术,植片应用鸵鸟脱细胞的角膜基质,术后分成两组,实验组术毕及术后结膜下注射复方倍他米松注射液(每隔7d一次),对照组术毕及术后结膜下注射地塞米松磷酸钠注射液(每隔7d一次)。分别于术后1、2wk, 1、2mo取兔角膜组织做石蜡包埋切片,进行HE染色观察组织特点,同时进行间接免疫荧光法检测CD4⁺、CD8⁺ T淋巴细胞的变化。

结果：术后2mo,实验组角膜植片在位,并保持透明,新生血管极少,组织切片HE染色和间接免疫荧光染色结果显示,仅见少许中性粒细胞浸润,未见CD4⁺、CD8⁺ T淋巴细胞; 对照组炎症反应明显,角膜植片混浊,组织切片HE染色和间接免疫荧光染色结果显示,炎性反应以中性粒细胞浸润为主,可见CD4⁺、CD8⁺ T淋巴细胞。

结论：复方倍他米松作为长效缓释剂可有效抑制鸵鸟-兔板层角膜移植术后免疫排斥反应,延长植片的存活时间。     

改良甘油长期冷冻保存角膜的超微结构及板层角膜移植

目的:研究改良甘油长期冷冻保存角膜的超微结构及板层角膜移植。方法:将涂有透明质酸钠(sodium hyaluronate,SH)的角膜片在-45℃以下长期甘油低温冷冻保存,保存12mo之后,用于15例板层角膜移植并进行透射电镜检查。结果:15例手术均顺利,术后随访6～24mo,13例植片透明,1例植片呈半透明状,1例植片部分新生血管化。透射电镜检查显示角膜超微结构保存良好。结论:甘油长期冷冻保存角膜有一定活性,适合应用板层角膜移植。     

Ex vivo transfer of Smad7 decreases damage to the corneal endothelium after penetrating keratoplasty

PURPOSE: To determine whether transient gene transfer and expression of the intracellular antagonist of transforming growth factor beta (TGF-beta), Smad7, to corneal endothelial cells decreases corneal endothelial cell damage after penetrating keratoplasty in a rabbit model. METHODS: Rabbit corneas were transfected ex vivo with replication-deficient adenoviruses encoding Flagtagged Smad7, Flag-tagged Smad3, or LacZ (termed AdCMV-Smad7, AdCMV-Smad3, AdCMV-LacZ) and then transplanted to normal rabbits. Expression of the exogenous Smads and phosphorylation of endogenous Smad2 in the transplanted corneal endothelium were examined by immunoblotting and immunohistochemistry with anti-Flag or anti-phosphorylated Smad2 antibodies. Cellular density and morphological changes in the corneal endothelium of the transplanted cornea were evaluated by scanning electron microscopy after transplantation of the Smad-transfected corneas. RESULTS: Transplanted AdCMV-Smad7-transfected corneas significantly inhibited the decrease in cellular density and accelerated wound healing at the host-graft junction when compared with transplanted AdCMV-LacZ-transfected corneas. Transplanted AdCMV-Smad3-transfected corneas showed decreased cellular density and delayed wound healing at the host-graft junction. CONCLUSIONS: Ex vivo gene transfer of Smad7 to corneal endothelial cells inhibits the decrease in cellular density and accelerates wound healing after penetrating keratoplasty in rabbits. Thus, modulation of Smad7 expression in corneal endothelial cells may decrease corneal endothelial cell damage after penetrating keratoplasty.     

Allogenic heterotopic cartilage transplantation for primary corneal replacement in a rabbit model

BACKGROUND: Aim of the present study was to investigate woundhealing after allogen heterotopic cartilage transplantation in a rabbit model. MATERIAL AND METHODS: Cartilage discs were transplanted by central lamellar keratoplasty (diameter 5.0 mm) in eleven New Zealand White Rabbits. Cartilage was harvested of the ears of rabbits at the day of transplantation. The cartilage discs 0.5-0.6 mm thick were prepared with 5.2 mm diameter and transplanted within 12 hours. Fixation was performed by eight 10-0 nylon sutures. Postoperatively, dexamethasone ointment containing gentamicin was applied three times a day for three weeks. Clinical course was documented by photographs. For histological examinations the transplanted eyes were enucleated after one day or three days, and after 3, 6 or 12 months. RESULTS: Allogen cartilage healed firmly in the cornea within two months. During the course of observation the cartilage grafts thinned and were replaced by normal appearing collagen fibrills. In one case neovascularisation of the cornea occurred and reached the cartilage graft, but did not invate. Histologically, in all cases no leukocytes could be seen in the cartilage or its neighbourhood. The cartilage grafts did not proliferate and remained avascular. The intraocular structures showed no inflammatory reaction. CONCLUSIONS: Allogen cartilage grafts to a clear corneal pounch do not induce an inflammatory reaction. Therefore, autologous cartilage prepared of ears seems to be useful biological material in ophthalmologic surgical procedures, for example to fill up corneal defects. To investigate if cartilage grafts will be helpful to stop neovascularisation in keratoplasty further studies with vascularisized corneas are necessary.     

Changing pattern of utilization of human donor cornea in India

Purpose:To review the changing pattern of donor, corneal utilization in an eye bank at a Tertiary Care Center in Northern India by analyzing the trend in the years 2003, 2008, and 2011.Methods:A retrospective review of eye bank records for 3 years (2003, 2008, and 2011) was performed at the National Eye Bank. Details including a clinical grade of donor cornea, indication of corneal transplantation (therapeutic or optical), type of procedure (penetrating or lamellar keratoplasty [LK]), and clinical diagnosis of the graft recipients were recorded. Primary outcome measure was to observe any preference toward LK, judicious usage of donor corneal tissue, and impact of lamellar corneal transplant in the usage of donor corneas. Secondary outcomes included overall utilization rate and change in trend of indication for keratoplasty.Results:A total of 673, 745, and 864 corneas were retrieved in the years 2003, 2008, and 2011, respectively. The percentage of donor corneal utilization increased significantly over time with the rate being 65.08%, 70.06%, and 68.29%, respectively, in the years 2003, 2008, and 2011 (P = 0.014); however, this change was reflected only in the usage of nonoptical grade corneas and not for the optical grade corneas. There was an overall increase in lamellar corneal procedures for any clinical grade of cornea (P = 0.0019); number of Descemet''s stripping automated endothelial keratoplasty (DSAEK) procedures increased significantly (P < 0.001), particularly for pseudophakic corneal edema (PCE) (P = 0.0085) and failed graft (P = 0.002). Significant increase in the utilization of nonoptical grade corneas was observed over the years (P = 0.005), though the utilization did not increase significantly for optical purposes viz., LK (P = 0.08).Conclusions:Utilization rate of donor corneas increased over the years, primarily due to increase in usage of nonoptical grade corneas for therapeutic purposes. There was a procedural shift toward DSAEK for PCE and failed graft. However, an increase in usage of nonoptical grade corneas for LK, a single donor corneal tissue for two recipients, and retrieval or utilization of optical grade cornea was not observed.     

A simple, cross-linked collagen tissue substitute for corneal implantation

PURPOSE: To develop a simple corneal substitute from cross-linked collagen. METHODS: Porcine type I collagen (10%; pH 5), was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). The final homogenous solution was molded to corneal dimensions, cured, and then implanted into rabbits and minipigs by lamellar keratoplasty. The implants were followed for up to 6 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, topography and esthesiometry for nerve function. Histopathologic examinations were also performed on rabbit corneas harvested after 6 months. RESULTS: Cross-linked collagen (refractive index, 1.35) had optical clarity superior to human corneas. Implanted into rabbit and porcine corneas, only 1 of 24 of the surgical corneas showed a slight haze at 6 months after surgery. All other implants showed no adverse reactions and remained optically clear. Topography showed a smooth surface and a profile similar to that of the contralateral nonsurgical eye. The implanted matrices promoted regeneration of corneal cells, tear film, and nerves. Touch sensitivity was restored, indicating some restoration of function. The corneas with implants showed no significant loss of thickness and demonstrated stable host-graft integration. CONCLUSIONS: Collagen can be adequately stabilized, using water soluble carbodiimides as protein cross-linking reagents, in the fabrication of corneal matrix substitutes for implantation. The simple cross-linking methodology would allow for easy fabrication of matrices for transplantation in centers where there is a shortage of corneas, or where there is need for temporary patches to repair perforations in emergency situations.     

组织工程角膜三维构建的实验研究

目的以人胚胎干细胞（hESC）诱导细胞为种子细胞,以脱细胞猪角膜基质（APCM）为支架三维构建生物工程角膜,以期用于穿透性角膜移植,解决角膜供体极度匮乏的难题。方法实验研究。无菌条件下将新鲜猪角膜组织置于0.5% SDS溶液中4 ℃脱细胞 24 h,获取APCM。将hESCs与人角膜基质细胞通过Transwell共培养5 d,获取眼周间充质干细胞（POMPs）,再于人晶状体上皮细胞源性条件培养基继续培养14 d获取角膜内皮样细胞并进行鉴定和筛选纯化。将纯化后扩增的角膜内皮样细胞接种于APCM构建角膜内皮植片,并移植入角膜内皮功能失代偿动物模型进行泵功能评估;采用人角膜缘干细胞（LSCs）来源的条件培养基培养hESCs 12 d,诱导其分化人角膜上皮样细胞并筛选鉴定,将其与APCM构建的角膜上皮植片移植于LSC失代偿动物模型的角膜缘,观察其眼表修复能力。结果诱导的人角膜内皮样细胞表达内皮细胞相关标记物vimentin、N-cadherin、Na+/K+ATP酶和ZO-1。构建的角膜内皮植片能够促使角膜内皮功能失代偿动物的角膜逐渐恢复透明。构建的角膜上皮细胞植片具有4～5层细胞复层结构,类似于正常角膜上皮,且能够一定程度上修复LSC失代偿动物模型眼表。结论采用hESCs诱导分化来源的细胞与APCM构建的人角膜内皮植片和人角膜上皮植片具有类似于正常角膜的功能,为全层生物角膜的构建提供了良好的实验和理论基础,具有良好的临床应用前景。     

Recurrent granular corneal dystrophy.

Four full-thickness corneal buttons and tissue from one lamellar keratoplasty, derived from four patients with granular corneal dystrophy recurring in previously transplanted corneas, were studied by light and transmission electron microscopy. Fibrous tissue without vascularization was present between the epithelium and Bowman's membrane and contained deposits characteristic of granular dystrophy in both light and electron microscopy. The recurrence of dystrophy in the normal donor cornea is the result of infiltration of the grafted cornea by host invasion. The donor stroma is spared.     

组织工程角膜研究和应用进展

中国角膜供体材料的严重缺乏导致众多的角膜盲患者不能通过角膜移植来复明仍是临床棘手的问题。近年来,细胞生物学、组织工程学和材料学的发展为替代人角膜材料开辟了更广阔的前景;而且以深板层移植和内皮移植为代表的成分板层移植的推陈出新从临床技术上有力地推动了组织工程角膜的研发。现就脱细胞角膜基质板层材料、上皮细胞和内皮细胞组织工程的基础研究和临床应用进展进行述评。     

Efficacy of pig-to-rhesus lamellar corneal xenotransplantation

PURPOSE. To solve the shortage of donor corneas, a decellularizing method based on hypertonic saline treatment was introduced, and a favorable outcome was observed in pig-to-rabbit lamellar corneal transplantation. This study was an investigation of the efficacy of pig-to-nonhuman primate lamellar corneal transplantation, using both decellularized and fresh porcine corneas to assess feasibility as a substitute for human corneas. METHODS. Nine Chinese rhesus macaques underwent lamellar corneal transplantation using both decellularized (n = 5) and fresh (n = 4) porcine corneas. Clinically acceptable graft size (7.5 mm in diameter) and minimal immunosuppression based on topical and systemic corticosteroids were applied. Rejection signs, histology of porcine grafts, and serial changes in recipients' blood profile, including memory T-cell subset, anti-α-Gal and donor pig-specific antibodies, and complement were evaluated. Changes in aqueous complement concentration were also assessed at 4 weeks after transplantation. RESULTS. Of the decellularized porcine lamellar grafts, 80% remained transparent for more than 6 months, whereas half of the fresh porcine lamellar grafts developed chronic rejection. Rejected grafts showed extensive cellular infiltration, predominantly CD8(+) T lymphocytes and macrophages. Immunologic profiles of the recipients with rejected grafts showed a significant increase in the concentration of aqueous complement, an enhancement of memory T cells, and an abrupt increase in donor pig-specific antibodies. CONCLUSIONS. The findings suggested that decellularized porcine cornea could be a promising substitute for human corneal allograft. Fresh porcine cornea may be a feasible option for a substitute if combined with more potent immunosuppression or if obtained from transgenic pigs with complement-regulatory proteins.     

Use of indocyanine green in deep lamellar endothelial keratoplasty

A new technique using indocyanine green (ICG) during deep lamellar endothelial keratoplasty (DLEK) to stain the corneal stroma of the donor disk facilitated surgical placement of the disk in the host corneal opening created to match the donor disk. Two female patients, aged 82 and 77 years, had ICG staining of the donor corneal disk during DLEK for pseudophakic bullous keratopathy and Fuchs' corneal dystrophy. By 24 hours postoperatively, no ICG was detected clinically by biomicroscopy of the sutureless (no corneal sutures) lamellar transplanted corneas. This is the first report of the use of ICG during DLEK and the first intrastromal use of ICG in the human cornea. The use of ICG facilitated the DLEK procedure and appears to be safe for intraoperative use in the cornea.     

用深低温保存的角膜材料行板层角膜移植术治疗边缘性角膜变性

目的:探讨用深低温保存的角膜材料行板层角膜移植术治疗边缘性角膜变性的效果。方法:对角膜边缘已经很薄,接近穿孔或已经穿孔的27例边缘性角膜变性患者,用深低温保存的角膜材料行板层角膜移植术治疗,观察术后视力,眼压,植片愈合及并发症等情况,随访时间为2a。结果:术后角膜边缘病变均未发展,术后1wk视力基本与术前相同,1.0者11例,0.6～0.8者6例,0.3～0.5者8例,0.05～0.25者2例。原穿孔的病例有术后一过性高眼压、前房反应、瞳孔欠圆等并发症。2a内只有3例发生了术后排斥反应。结论:用深低温保存的角膜材料完全可以用来进行板层角膜移植治疗边缘性角膜变性,弥补了新鲜角膜的来源限制与时效限制,是治疗边缘性角膜变性的理想材料。     

诱导人脐带间充质干细胞分化为角膜上皮样细胞的研究

背景眼表疾病导致的角膜盲已成为全球致盲性角膜疾病中的主要原因之一。随着组织工程技术的发展和进步,组织工程角膜为眼表疾病的治疗开辟了新的途径。目的观察体外培养的人脐带间充质干细胞（UC—MSCs）移植到兔角膜基质后的分化发育情况,探讨人UC-MSCs分化为角膜上皮细胞以及治疗兔角膜损伤的可行性。方法获取人脐带组织,采用Ⅳ型胶原酶消化法分离纯化人UC—MSCs并传代,取第3代细胞用于扩增和实验。流式细胞仪检测细胞的免疫表型及诱导成骨分化鉴定。24只新西兰大白兔按随机数字表法随机分为2个组,将人UC—MSCs接种于去上皮的猪角膜基质上,培养4d后行实验组兔左眼板层角膜移植;对照组以相同的手术方法单纯移植去上皮猪角膜基质。术后对角膜定期行活体激光共焦显微镜检查,并分别于术后2、4、8周摘除各组实验眼行组织病理学和免疫荧光检查,评价移植到兔角膜基质的人UC-MSCs的存活、分化以及移植局部的反应等;应用免疫荧光技术检测移植后角膜上皮细胞中角蛋白3（CK3）、CKl2以及转运蛋白G超家族成员（ABCG2）的表达。结果消化培养的人UC—MSCs呈圆形,细胞胞体较大,贴壁后细胞呈长梭形。培养获得的人UC—MSCs的细胞表型CD105^＋／CD29^＋／CD44^＋／CD34^-／CD45^-,并可诱导分化为成骨细胞。实验组人ISC—MSCs接种到去上皮猪角膜基质后贴附良好、生长迅速,术后植片在植床上存活良好,种植了人UC-MSCs的去上皮猪角膜移植到兔眼,可见实验组受体角膜较对照组透明,未见明显新生血管,在活体共焦显微镜下可见新生的角膜上皮样细胞,未发生免疫排斥反应。免疫荧光检测可见在重建的角膜上皮层检测到CK3及CKl2的阳性表达,而未见ABCG2的表达。结论将种植了人UC—MSCs的猪角膜基质移植到损伤的兔角膜后,人UC—MSCs可以存活、增生并分化为角膜上皮样细胞,可用于修复甚至重建损伤的角膜表层。