Small-diameter acellular porcine corneal stroma for peripheral corneal ulceration treatment

AIM:To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma(SAPS)for the treatment of peripheral corneal ulceration(PCU).METHODS:This retrospective clinical study included 18 patients(18 eyes)with PCU between April 2018 and December 2020.All patients had PCU and underwent lamellar keratoplasty with SAPS.Observation indicators included preoperative and postoperative best-corrected visual acuity(BCVA)and transparency of SAPS.The infection control rate in the surgical eye-lesion area was also calculated.RESULTS:Eighteen patients underwent lamellar keratoplasty with SAPS to treat PCU.None of the patients experienced rejection after 6mo(18/18)and 12mo(16/16)of follow-up.The BCVA(0.47±0.30)at the 6mo followup after operation was significantly improved compared with the baseline(0.99±0.80),and the difference was statistically significant(Z=-3.415,P<0.05).The BCVA at the 12mo follow-up after operation was not statistically significant compared to the 6mo(Z=0,P=1).With time,the SAPS graft gradually became transparent.At the 6mo(18/18)and 12mo(16/16)follow-up,none of the patients had recurrent corneal infection.CONCLUSION:SAPS is clinically effective in the treatment of PCU,improving the patient’s BCVA and reducing the incidence of rejection after keratoplasty.

Establishment of an untransfected human corneal stromal cell line and its biocompatibility to acellular porcine corneal stroma

AIM: To establish an untransfected human corneal stromal (HCS) cell line and characterize its biocompatibility to acellular porcine corneal stoma (aPCS). METHODS: Primary culture was initiated with a pure population of HCS cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various necessary growth factors. The established cell line was characterized by growth property, chromosome analysis, tumorigenicity assay, expression of marker proteins and functional proteins. Furthermore, the biocompatibility of HCS cells with aPCS was examined through histological and immunocytochemistry analyses and with light, electron microscopies. RESULTS: HCS cells proliferated to confluence 2 weeks later in primary culture and have been subcultured to passage 140 so far. A continuous untransfected HCS cell line with a population doubling time of 41.44 hours at passage 80 has been determined. Results of chromosome analysis, morphology, combined with the results of expression of marker protein and functional proteins suggested that the cells retained HCS cell properties. Furthermore, HCS cells have no tumorigenicity, and with excellent biocompatibility to aPCS. CONCLUSION: An untransfected and non-tumorigenic HCS cell line has been established, and the cells maintained positive expression of marker proteins and functional proteins. The cell line, with excellent biocompatibility to aPCS, might be used for in vitroreconstruction of tissue-engineered HCS.

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.·R ESULTS：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 vitro.

Construction of a full-thickness human corneal substitute from anterior acellular porcine corneal matrix and human corneal cells

AIM: To construct functional human full-thickness corneal replacements.METHODS: Acellular porcine corneal matrix(APCM) was developed from porcine cornea by decellulariztion. The biomechanical properties of anterior-APCM(AAPCM) and posterior-APCM(PAPCM) were checked using uniaxial tensile testing. Human corneal cells were obtained by cell culture. Suspending ring was designed by deformation of an acupuncture needle. MTT cytotoxicity assay was used to check the cytotoxicity of suspending ring soaking solutions. A new three-dimensional organ culture system was established by combination of suspending ring, 48-well plate and medium together. A human full-thickness corneal substitute was constructed from human corneal cells with AAPCM in an organ coculture system. Biochemical marker expression of the construct was measured by immunofluorescent staining and morphological structures were observed using scanning electron microscopy. Pump function and biophysical properties were examined by penetrating keratoplasty and follow-up clinical observations.RESULTS: There were no cells in the AAPCM or PAPCM, whereas collagen fibers, Bowman's membrane, and Descemet's membrane were retained. The biomechanical property of AAPCM was better than PAPCM. Human corneal cells grew better on the AAPCM than on the PAPCM.There was no cytotoxicity for the suspending ring soaking solutions. For the constructed full-depth human corneal replacements keratocytes scattered uniformly throughout the AAPCM and expressed vimentin. The epithelial layer was located on the surface of Bowman's membrane and composed of three or four layers of epithelial cells expressing cytokeratin 3. One layer of endothelial cells covered the stromal surface of AAPCM, expressed Na+/K+ATPase and formed the endothelial layer. The construct was similar to normal human corneas, with many microvilli on the epithelial cell surface, stromal cells with a long shuttle shape, and zonula occludens on the interface of endothelial cells. The construct withstood surgical procedures during penetrating keratoplasty. The corneal transparency increased gradually and was almost completely restored 7 d after surgery.CONCLUSION: AAPCM is an ideal scaffold for constructing full-thickness corneal replacement, and functional human full-thickness corneal replacements are successfully constructed using AAPCM and human corneal cells.

Acellular ostrich corneal stroma used as scaffold for construction of tissue-engineered cornea

AIM： To assess acellular ostrich corneal matrix used as a scaffold to reconstruct a damaged cornea. METHODS： A hypertonic saline solution combined with a digestion method was used to decellularize the ostrich cornea. The microstructure of the acellular corneal matrix was observed by transmission electron microscopy （TEM） and hematoxylin and eosin （H＆E） staining. The mechanical properties were detected by a rheometer and a tension machine. The acellular corneal matrix was also transplanted into a rabbit cornea and cytokeratin 3 was used to check the immune phenotype, RESULTS： The microstructure and mechanical properties of the ostrich cornea were well preserved after the decellularization process, in vitro, the methyl thiazolyl tetrazoUum results revealed that extracts of the acellular ostrich corneas （AOCs） had no inhibitory effects on the proliferation of the corneal epithelial or endothelial cells or on the keratocytes, The rabbit lamellar keratoplasty showed that the transplanted AOCs were transparent and completely incorporated into the host cornea while corneal turbidity and graft dissolution occurred in the acellular porcine cornea （APC） transplantation, The phenotype of the reconstructed cornea was similar to a normal rabbit cornea with a high expression of cytokeratin 3 in the superficial epithelial cell layer, CONCLUSION： We first used AOCs as scaffolds to reconstruct damaged corneas. Compared with porcine corneas, the anatomical structures of ostrich corneas are closer to those of human corneas. In accordance with the principle that structure determines function, a xenograft lamellar keratoplasty also confirmed that the AOC transplantation generated a superior outcome compared to that of the APC graft.

脱细胞猪角膜基质深板层移植治疗感染性角膜炎的疗效观察

目的:评价脱细胞猪角膜基质深板层移植治疗感染性角膜炎的有效性、安全性。方法:前瞻性研究。收集2017-02/2020-10在我院经药物保守治疗无效的感染性角膜炎患者17例17眼,应用脱细胞猪角膜基质进行深板层角膜移植手术。术后随访6 mo,观察BCVA、角膜上皮愈合情况、角膜透明情况,并记录有无感染复发以及植片排斥。结果:所有患者中均顺利完成深板层角膜移植手术,均未发生术中并发症,无失访病例,术后角膜感染均得到有效控制。术后BCVA均较术前改善(P<0.05)。17眼中16眼术后1 mo上皮完全覆盖角膜植片,仅1眼上皮未完全愈合,为病毒性角膜炎的反复发作。术后1.5 mo所有患者上皮均完全愈合。术后1 mo角膜水肿开始改善,其中1眼角膜植片轻度混浊,16眼中重度混浊;术后3-6 mo角膜透明度稳定,至术后6 mo时4眼完全透明,6眼轻度混浊,6眼中度混浊,1眼重度混浊。随访过程中,无感染复发,有1眼发生眼压升高现象,经治疗后眼压恢复正常。结论:脱细胞猪角膜基质深板层移植治疗感染性角膜炎,不仅能有效控制感染,缓解患者角膜刺激症状,恢复角膜的解剖与功能,而且能维持良好的透明性,提高患者视力,可作为替代同种异体人角膜。

生物工程角膜板层角膜移植在真菌性角膜溃疡患者中的应用

目的探讨生物工程角膜板层角膜移植在真菌性角膜溃疡患者中的有效性及安全性。方法选取44例真菌性角膜溃疡患者为研究对象,根据使用的角膜材料分为生物工程组[采用脱细胞猪角膜基质(APCM)进行板层角膜移植,20例]和人供体组(采用人供体角膜材料进行板层角膜移植,24例)。比较两组术后不同时间角膜植片透明度评分、最佳矫正视力(BCVA)和术后角膜上皮愈合时间、术后并发症及复发率。结果术后1、3、6、12个月,随着时间的延长两组角膜植片透明度评分均逐渐降低,术后1、3、6个月生物工程组角膜植片透明度评分高于人供体组(P<0.05),但术后12个月两组角膜植片透明度评分比较,差异无统计学意义(P>0.05)。术后3、6、12个月,随着时间的延长两组患眼BCVA均逐渐增加,但生物工程组患眼术后3、6、12个月的BCVA均低于人供体组(P<0.05);两组角膜上皮愈合时间及术后角膜上皮愈合延迟、角膜植片排斥、角膜植片溶解、新生血管长入角膜植片4项并发症发生率和术后复发率比较,差异无统计学意义(P>0.05)。结论以APCM为材料进行板层角膜移植治疗真菌性角膜溃疡临床效果好,安全性高,可作为生物工程角膜应用于角膜病致盲的治疗。

生物角膜用于人角膜板层移植术后的临床疗效及激光扫描共焦显微镜动态观察

背景近年来由于角膜供体材料的缺乏，异种角膜移植研究逐渐受到研究者的关注，猪角膜脱细胞基质构建的生物角膜支架具有良好的生物相容性并已制备成生物角膜材料，但其用于人眼角膜移植后的临床效果及其组织形态学变化特征尚不清楚。目的观察用生物角膜进行人板层角膜移植术后的临床效果，并利用激光扫描共焦显微镜进行活体动态观察，评估生物角膜作为新型角膜替代材料进行人眼角膜板层移植的可行性。方法采用前瞻性系列病例观察研究方法，于2014年2—8月在河南省立眼科医院纳入药物治疗无效的感染性角膜炎或陈旧性角膜白斑患者15例15眼，包括真菌性角膜炎4例，细菌性角膜炎1例，混合感染性角膜炎9例，陈旧性角膜白斑1例。采用猪角膜脱细胞基质构建的生物角膜行患眼生物角膜板层移植术，以术眼的对侧正常眼作为对照。分别于术后3d、7d、1个月、3个月、6个月、9个月和12个月对术眼进行随访，对植片愈合情况进行评分，随访时间为1年。疗效指标主要包括角膜透明度、新生血管形成、角膜植片有无溶解或感染复发、最佳矫正视力（logMAR）。分别于术后3、6、9和12个月利用激光扫描共焦显微镜对角膜进行动态观察，记录和分析移植后角膜上皮和内皮细胞的形态和密度、角膜基质的形态特征以及角膜上皮下神经纤维的恢复情况。结果术眼术后3d早期出现角膜水肿等炎症反应，术后7d炎症反应消退。术后1个月植片透明，随访期间未出现角膜溶解、感染复发现象。术后6个月时1例患者出现青光眼，术后12个月时1例患者发生排斥反应。术眼术前及术后不同时间点间logMAR、角膜透明度评分、角膜新生血管评分的总体比较差异均有统计学意义（X2=92．63、59．37、10．50，均P〈0．05），其中术后视力、角膜透明度均较术前明显改善，差异均有统计学意义（均P〈0．05）。激光扫描共焦显微镜检查结果显示，术眼术后受体角膜上皮细胞逐渐覆盖植片，完成正常的上皮化，角膜上皮细胞形态接近对侧正常眼，但角膜内皮细胞体积大于对侧眼，而植片角膜基质中无细胞生长，明显与对侧眼角膜基质不同；术后6个月时8例患者植片内有上皮下神经纤维长入，但神经纤维走形弯曲，密度明显低于对侧正常眼。术后不同时间点角膜上皮细胞密度总体比较差异均无统计学意义（F=1．48，P=0．22）。对侧正常眼、术眼术后3、6、9和12个月角膜内皮细胞密度分别为（2542±119）、（1895±129）、（1869±135）、（1854±101）和（1844±103）个／mm2，术眼术后各个时间点角膜内皮细胞密度均明显低于对侧正常眼，差异均有统计学意义（均P〈0．05）；术后6、9和12个月角膜上皮下神经纤维密度值分别为（1．26±0．13）、（3．62±0．81）和（5．98±0．44）mm／mm2，均明显高于前1个时间点的测定值，差异均有统计学意义（t′=-8．16、-7．24，均P=0．00）。结论用生物角膜行人眼角膜板层移植术后植片即可长期存活，重建眼表结构，改善患者视力，可替代人角膜供体材料。

脱细胞猪角膜基质体外支持角膜上皮和基质细胞的生长(英文)

目的：探讨脱细胞猪角膜基质体外能否支持兔角膜细胞的生长。方法：体外培养兔角膜上皮细胞和基质细胞,并接种到制备的脱细胞猪角膜基质上,倒置相差显微镜和组织学观察细胞生长情况。结果：上皮细胞能在脱细胞猪角膜基质上贴附生长,10d时可形成2～3层的复层结构。基质细胞在脱细胞猪角膜基质上贴附生长后可向材料深层迁徙。结论：制备的脱细胞猪角膜基质体外可支持兔角膜上皮细胞和基质细胞的生长。

异种角膜基质的研究现状及问题

在角膜3层(上皮层+Bowman’s膜、基质层、内皮层+Descemet’s膜)膜中,按等量计算,基质层的免疫原性是最低的。应用脱细胞的异种角膜基质移植后,神经和基质细胞可以再生,术后未见排斥反应,植片获得透明。因此,异种角膜基质有望成为板层角膜移植的供体和组织工程角膜的载体。现就异种角膜基质的研究现状及问题综述如下。

脱细胞真皮基质在角膜中植入性的生物力学研究

对脱细胞真皮基质(acellular dermal matrix,ADM)囊袋植入兔角膜后的生物相容性、生物学转归及组织结构改变进行生物力学研究,以探讨其作为组织工程角膜支架材料的可行性.对ADM进行了系统的组织微观结构观察和植入性实验研究,组织结构观察包括HE染色、胶原染色、免疫荧光染色和透射电镜观察.在植入实验中,将ADM按不同预处理方法分为无干预组、脱水组和复水组,将3组ADM囊袋植入兔角膜,观察其术后不同时期在体和离体的生物相容性和组织学特性.结果发现:ADM由横纵交替、排列疏松的Ⅰ和Ⅲ型胶原纤维束构成,未见细胞结构;囊袋植入角膜后有不同程度的炎症反应,部分植片透明;组织学观察显示3组ADM植入角膜后均有细胞长入,并进行了胶原纤维重塑.研究工作表明,经适当预处理且结构致密的脱细胞真皮基质植入后可以长期稳定存留于角膜内,是一种比较理想的组织工程角膜支架.

生物工程角膜移植对真菌性角膜溃疡的临床疗效及其对组织形态学的影响

目的探讨生物工程角膜移植对真菌性角膜溃疡的临床疗效及对组织形态学的影响。方法选取2017年10月至2019年5月遂宁市中心医院收治的30例真菌性角膜溃疡患者为研究对象。采用脱细胞猪角膜基质构建的生物角膜对患眼进行角膜板层移植术,以对侧眼为对照。随访12个月,观察移植后移植物上皮化完成情况,利用共聚焦显微镜观察移植物角膜上皮细胞、内皮细胞密度和上皮下神经纤维修复情况。结果术后随访中患者均未再发现孢子和菌丝,术后1年,术眼角膜表面曲线光滑度与对侧眼相当。术后角膜上皮愈合时间为3 d至1周,随时间延长,患者眼部刺激性症状评分、新生血管评分、移植物透明度评分均呈下降趋势。术后3、6、12个月,术眼上皮细胞密度与对侧眼比较差异均无统计学意义(均P>0.05)。术后3、6、12个月,术眼内皮细胞密度均显著低于对侧眼(均P<0.05)。术后6个月,术眼上皮下神经纤维密度最低(P<0.05),术后12个月,术眼上皮下神经纤维密度逐渐增加,但仍显著低于对侧眼(P<0.05)。结论采用生物工程角膜行板层角膜移植治疗真菌性角膜溃疡的临床效果显著,安全性高,患者预后较好。

猪脱细胞角膜基质的制备及组织学特征观察

目的:制备低抗原性、保留天然角膜结构的组织工程角膜支架材料。方法:对猪角膜进行反复冻融联合酶消化,最后冷冻干燥,观察其组织学特征。结果:制备的脱细胞猪角膜基质的细胞成分能被有效去除,材料保留了利于角膜上皮生长的基底膜和有序排列的网状的基质胶原结构,有一定的强度和韧性。结论:采用反复冻融联合酶消化、冷冻干燥法能获得一种无细胞的组织工程角膜支架材料。

脱细胞猪角膜基质体外支持皮肤细胞生长的实验研究

目的探讨脱细胞猪角膜基质体外是否支持皮肤细胞的生长。方法制备脱细胞猪角膜基质(前期实验已完成)。体外培养人皮肤表皮细胞和成纤维细胞,取第3代人皮肤成纤维细胞接种在脱细胞猪角膜基质的中间基质面,培养3d后,将材料翻转,取第3代人皮肤表皮细胞接种在材料的上皮面上,再培养10d后,取细胞-支架复合体制作石蜡切片,HE染色后光镜下观察。结果组织学观察显示皮肤的表皮细胞和成纤维细胞体外均可在脱细胞猪角膜基质上黏附生长。接种10d后,皮肤表皮细胞在材料表面形成复层结构,可见角化的细胞。接种13d可见成纤维细胞存活并在支架层间生长。结论脱细胞猪角膜基质有良好的细胞相容性,在体外能支持皮肤细胞的生长和增生。

用共聚焦显微镜观察角膜原位磨镶术术后角膜基质中“低细胞区”形态学改变

目的角膜原位磨镶术(Laserin situ keratom ileusis,LASIK)通过制作角膜瓣,在其下基质中用激光切削组织,改变角膜曲率,达到矫正屈光不正的目的。文中旨在应用共聚焦显微镜探讨LASIK术后远期角膜基质中"低细胞区域"的相关因素。方法设计自身配对实验研究。选择2003年4月至2009年7月期间在天津医科大学眼科中心屈光手术门诊和武警医学院附属医院眼科就诊LASIK手术的病例34人(68眼),按等效屈光度分为2组,屈光度<-6.0D为低、中度近视眼组37眼,屈光度>-6.0D为高度近视眼组31眼。分别在术前、术后第1、3、7、10、30、60、90天,应用活体共聚焦显微镜(Con-foscan 3.0)观察中央角膜基质细胞形态学改变,测量角膜各层厚度并进行相关性分析。结果 68眼(100%)中央角膜见到平均深度为(44.01±10.67)μm的低细胞区域。低细胞区内的角膜细胞核排列无序,细胞核形态与正常角膜细胞核有异,细胞核反射亮度正常,细胞缺失区呈片状。低、中度近视眼与高度近视眼的界面角膜细胞核数、低细胞区域深度差异无统计学意义。结论角膜原位磨镶术后远期角膜基质出现"低细胞区",分析低细胞区的深度与手术时间、年龄、术前屈光状态、激光削切深度、激光能量、角膜瓣基质厚度、角膜瓣厚度、剩余基质床厚度、上皮厚度以及均无相关性。。

飞秒激光辅助脱细胞猪角膜移植术治疗真菌性角膜炎的护理

目的 探讨飞秒激光辅助脱细胞猪角膜基质（APCS）行板层角膜移植术治疗真菌性角膜炎的护理方法及效果。方法 对临床确诊为真菌性角膜炎的37例（37眼）患者用APCS行板层角膜移植术,同时给予针对性护理和定期随访。结果 全部病例术后随访4~6个月,31例（83.8%）患者较术前视力提高了3行以上,6例（16.2%）患者视力保持不变;37例患者术后眼部刺激症状均得到缓解;所有病例角膜植片均在7d内全部上皮化,角膜植片术后2周变透明,随访期间均无真菌感染复发征象。结论 术前加强心理护理和固视训练,术后密切病情观察和眼部护理,出院前进行科学指导和示范滴眼,出院后做好定期随访和用药监督,是巩固治疗成果的重要环节。

猪脱细胞角膜基质人板层角膜移植术后植片上皮化的临床观察

目的:观察猪脱细胞角膜基质进行人板层角膜移植术后角膜植片上皮化的临床过程。方法:收集2017年1月至2018年1月在佛山市第二人民医院经药物治疗无效的感染性角膜炎患者5例。采用脱细胞角膜基质(商品名:艾欣瞳)行板层移植术,以对侧眼为对照。术后3 d,7 d,1个月,3个月,6个月对患者进行随访,在裂隙灯显微镜下观察移植后植片上皮化完成过程。术后1,3,6个月共聚焦显微镜观察植片上角膜上皮细胞密度及上皮下神经纤维修复情况。结果:脱细胞角膜基质板层角膜移植术后3 d开始出现上皮化,术后10d上皮化逐步完成,但角膜上皮排列疏松,荧光素染色仍有点状染色。激光扫描共聚焦显微镜检查结果显示:术后1,3,6个月角膜上皮细胞排列规则、紧密,与对侧眼比较角膜上皮细胞密度差异无统计学意义(P>0.05)。结论:应用脱细胞角膜基质行板层角膜移植术后植片上皮化过程能够顺利完成。

生物工程角膜治疗真菌性角膜炎的临床效果

目的观察生物工程角膜治疗真菌性角膜炎的临床效果。方法选取15例(15眼)真菌性角膜炎患者,均使用脱细胞猪角膜基质构建的生物工程角膜施行板层角膜移植手术治疗。观察患者术后早期(术后1个月)恢复情况、术后远期效果及并发症发生情况,比较患者手术前后的视力。结果随访期间,所有患者眼部感染均得到控制,无感染复发,自觉症状均在术后1~2周内逐渐改善。除1例角膜植片在术后2个月发生溶解以外,其余患者的生物角膜维持在基本透明的状态,视力均有不同程度的提高。结论板层角膜移植术中采用生物工程角膜治疗真菌性角膜炎安全、有效。

小鼠胚胎干细胞条件培养液培养的人角膜内皮细胞在脱细胞猪角膜基质上单层细胞片的构建

背景 目前角膜供体来源短缺,且体外培养的人角膜内皮细胞（HCECs）难以再生和扩增,为临床上角膜移植的开展带来了很大困难,组织工程角膜的构建仍是研究的主要课题.前期研究已证实,小鼠胚胎干细胞条件培养基（ESC-CM）能够促进体外HCECs的增生,脱细胞猪角膜基质（APCS）是较好的支架材料,但ESC-CM培养的HCECs是否能在APCS上融合成单层细胞鲜见研究. 目的 研究ESC-CM培养的HCECs在APCS上是否能够形成单层细胞. 方法 用小鼠ESC-CM培养液培养小鼠ES-E14细胞,收集培养上清液,离心后按体积比1∶3与人角膜内皮细胞培养液（CEM）混合,制备体积分数25％ ESC-CM液.取穿透角膜移植术后剩余的供体人角巩膜缘组织,采用组织块培养法用ESC-CM对HCECs进行培养和传代,采用逆转录PCR法检测HCECs中Ⅷ型胶原蛋白（ColⅧ）与神经元特异性烯醇化酶（NSE）的表达以鉴定细胞.取猪角膜,利用磷脂酶A2和碳酸氢盐溶液脱细胞法制备APCS,将第2代HCECs混合液按照800/mm2的密度种植于灭菌的APCS上进行培养,于倒置相差显微镜下观察细胞形态,采用苏木精-伊红染色法观察培养细胞的组织结构;采用免疫荧光法检测构建的HCECs单细胞片中闭锁小带蛋白-1（ZO-1）和Na＋-K＋-ATP酶的表达. 结果 体外培养的HCECs呈典型的六角形,内皮特异性标志物ColⅧ和NSE mRNA表达阳性.脱细胞APCS呈白色透明状,苏木精-伊红染色显示APCS中无角膜细胞,但角膜胶原纤维排列规则.第2代HCECs复水后可在APCS上生长并贴附,培养后7d融合成单层细胞片,细胞片上HCECs的细胞密度为（2 694± 143）/mm2.构建的HCECs片中内皮细胞泵功能相关蛋白ZO-1和Na＋-K＋-ATP酶均呈阳性表达,呈红色荧光. 结论 25％ESC-CM可促进HCECs的生长并维持细胞的正常形态,APCS可为HCECs片的构建提供支架和较好的生存微环境.在APCS形成的HCECs单细胞片可表达HCECs泵功能,是角膜移植的良好供体.

人脐静脉内皮细胞移植治疗角膜内皮功能衰竭的动物实验研究

目的:探讨异种脱细胞角膜基质为载体培养人脐静脉内皮细胞(HUVEC)进行后板层角膜移植(PLEK)治疗角膜内皮衰竭的可行性。方法:新西兰白兔30只,随机分为3组,实验组、基质组和对照组,每组10只,术中去除角膜内皮细胞,建立角膜内皮衰竭动物模型,实验组和基质组行后板层角膜移植术,对照组仅去除角膜后板层组织,不进行移植。术后观察3mo,对三组角膜的水肿混浊程度和中央角膜厚度进行统计学分析。结果:术后7d,实验组角膜水肿程度较基质组和对照组明显减轻,透明度增加。术后3mo时,实验组内皮细胞密度为2026.4±129.3个/mm2,中央角膜厚度平均为505.2±25.4μm,基质组中央角膜厚度平均为1 535.6±114.5μm,而对照组为1493.5±70.2μm。结论:实验以异种脱细胞角膜基质为载体培养人脐静脉内皮细胞,行后板层角膜移植术,治疗角膜内皮衰竭取得了初步成功。移植的人脐静脉内皮细胞能够在活体上成活,并具有一定的角膜内皮细胞的生物学功能,维持角膜透明,为临床上治疗角膜内皮疾病提供了新的思路和方法。