角膜内皮细胞治疗研究进展

人角膜内皮细胞(HCECs)是一种有丝分裂后的单层内皮细胞,因此,其在体内和体外的增殖能力十分有限。HCECs在严重受损的情况下会发生内皮失代偿,极易引起失明。目前,唯一有效的治疗方法是使用含健康角膜内皮的供体植片进行角膜移植。因此,世界范围内供体材料的严重短缺推动了对角膜内皮替代来源的研究。随着HCECs的细胞培养研究的不断开展,细胞治疗为角膜内皮失代偿提供了希望。本文对角膜内皮细胞治疗方面的最新研究进展进行综述。     

刀豆蛋白A条件化培养基对体外培养兔角膜内皮细胞Na^＋-K^＋-ATP酶的作用

目的 研究刀豆蛋白A(ConA)条件化培养基对体外培养兔角膜内皮细胞膜表面Na+-K+-ATP酶的分布及其酶活性的影响.方法 超微量ATP酶试剂盒测定原代培养兔角膜内皮细胞细胞膜表面Na+-K+-ATP酶的活性,并对ConA条件化培养基作用下不同生长周期(5、7、10 d)细胞Na+-K+-ATP酶的活性进行测定;免疫酶组织化学电镜下观察不同培养基作用下体外培养角膜内皮细胞膜表面Na+-K+-ATP酶的分布.结果 ConA条件化培养基组兔角膜内皮细胞Na+-K+-ATP酶活性明显高于无ConA组(P＜0.01),原代细胞生长7～10 d时Na+-K+-ATP酶活性最高(P＜0.01).单层兔角膜内皮细胞膜外侧可见Na+-K+-ATP酶阳性反应.在ConA条件化培养基处理组,其阳性反应物较多、致密;而在无ConA组则阳性反应物较少、疏松.结论 体外原代培养角膜内皮细胞在7～10 d时,Na+-K+-ATP酶活性最高.ConA条件化培养基可提高兔角膜内皮细胞Na+-K+-ATP酶活性.     

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.     

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.     

湿房和深低温保存角膜穿透性移植术后角膜内皮分析

目的　探讨深低温长期保存和短期湿房保存角膜移植术后植片角膜内皮细胞形态学特征。方法　应用深低温长期保存 30～ 5 0个月 (平均 2 5 .0± 11.9个月 )青壮年供体眼角膜完成穿透性角膜移植 2 4例 (2 4只眼 ) ,同时与来自于青壮年供体经 4℃湿房保存的新鲜供眼角膜穿透性移植术后的角膜内皮细胞状况相比较。全部患者术后角膜植片保持透明 ,未曾出现移植免疫排斥反应 ,手术操作由同一医生完成。术后 2～ 2 0 (平均 6 .7± 3.1)个月应用接触式角膜内皮显微镜及与其同步显示的角膜内皮细胞分析仪对角膜植片中央内皮细胞进行观察和分析。结果　应用深低温保存和湿房保存角膜行穿透性角膜移植术后角膜内皮细胞平均面积、细胞平均密度、面积变异系数、细胞最大面积、细胞最小面积以及六角形细胞出现率间均无显著性差异 (t=0 .116～ 1.195 ,P >0 .0 5 )。结论　应用深低温保存角膜和湿房保存角膜行穿透性角膜移植术后角膜内皮细胞具有相同的临床价值     

脱水保存角膜基质为载体培养角膜内皮细胞的实验研究

目的探讨以脱水保存角膜基质/后弹力层为载体培养角膜内皮细胞,构建组织工程化角膜内皮细胞移植膜的可行性及其机理。设计实验性研究。研究对象体外培养的兔角膜内皮细胞和脱水保存角膜基质/后弹力层。方法兔角膜经中性蛋白酶37°C孵育5min,去除内皮细胞保留后弹力层和角膜基质,无水氯化钙脱水后低温保存,使用前磷酸盐缓冲液复水。纯化的角膜内皮细胞接种于基质载体的后弹力层上进行体外培养,直至生长融合为细胞单层,倒置显微镜下观察细胞形态学变化。在不同时间点(1、2、4、6d)收集植片进行HE染色和电镜检测,分析组织结构的变化。主要指标角膜内皮细胞在脱水保存角膜基质/后弹力层载体上形成单层时间和生长特性,组织工程化角膜内皮细胞移植膜的三维结构和超微结构。结果角膜内皮细胞在载体上快速贴壁生长并增殖,体外培养6～7d即融合成单层,复合角膜内皮组织由基质/后弹力层和单层扁平内皮细胞组成,与生理状态下的角膜内皮组织相近。电镜下组织培养的兔角膜内皮细胞间连接紧密,细胞为多边形,胞核清晰,具有正常兔角膜内皮细胞的超微结构。结论角膜内皮细胞能够在干燥脱水保存基质/后弹力层载体上良好生长,并形成形态结构与正常角膜内皮组织相似的细胞单层,为角膜内皮细胞移植提供了新的载体选择。(眼科,2006,15:164-168)     

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。     

ResolvinE1对高危角膜移植免疫排斥反应的抑制作用

背景 以往防治角膜移植术后排斥反应的药物存在诱发局部或全身不良反应的风险,研究表明resolvinE1(RyE1)能够调节辅助性T细胞1(Th1)型免疫反应,但其对高危角膜移植术后的植片排斥反应有无抑制作用尚不清楚. 目的 观察高危角膜移植动物模型局部应用RvE1对植片免疫排斥反应的抑制作用.方法 以BALB/c小鼠为受体、C57BL/6小鼠为供体行角膜移植术.采用随机数字表法将90只BALB/c小鼠随机分成异体角膜移植组、异体角膜移植+RvE1组和自体角膜移植组,每组各30只.BALB/c小鼠右眼先用缝线法刺激2周以建立高危角膜移植眼模型,然后行穿透角膜移植术.异体角膜移植组和异体角膜移植+RvE1组小鼠右眼行异体角膜移植,自体角膜移植组小鼠将右眼角膜植片旋转180°后缝合于植床上.异体角膜移植组及自体角膜移植组小鼠术后每日用生理盐水10μl结膜下注射1次,异体角膜移植+RvE1组小鼠术后同法注射终质量浓度为0.1 μg/μl的RvE1 10μl,连续7d.术后裂隙灯显微镜下观察小鼠角膜植片反应并对其排斥反应进行评分.术后21 d处死各组小鼠各20只,收集小鼠术眼角膜、眼球和术眼侧颈部淋巴结,采用苏木精-伊红染色法观察各组小鼠角膜植片的组织病理学变化;采用免疫组织化学法检测术眼角膜中CD4及γ干扰素(IFN-γ)的表达;采用流式细胞术检测术眼侧颈部淋巴结淋巴细胞中Th1细胞(CD3+ CD8a-IFN-γ+)比例;采用荧光定量PCR法检测Th1细胞相关因子白细胞介素-2(IL-2)、肿瘤坏死因子-α(TNF-c)、IFN-γ及T-bet mRNA的相对表达水平.结果 异体角膜移植+RvE1组小鼠角膜植片存活时间为(28.5±1.7)d,明显长于异体角膜移植组的(14.0±1.6)d,差异有统计学意义(t=4.14,P＜0.001),自体角膜移植组小鼠植片在术后50 d存活率为100％.苏木精-伊红染色显示,异体角膜移植+RvE1组和自体角膜移植组小鼠角膜植片水肿及炎性细胞浸润程度均轻于异体角膜移植组.免疫组织化学法检测显示,各组角膜全层均有CD4表达,而IFN-γ主要表达于角膜上皮层,异体角膜移植组小鼠角膜组织中CD4和IFN-γ阳性细胞数均明显多于异体角膜移植+RvE1组和自体角膜移植组.流式细胞术检测显示,异体角膜移植+RvE1组和自体角膜移植组小鼠淋巴细胞中Th1细胞比例分别为(1.07±0.25)％和(0.85±0.12)％,明显低于异体角膜移植组的(1.56±0.20)％,差异均有统计学意义(均P＜0.05).荧光定量PCR检测显示,异体角膜移植组小鼠角膜中IL-2、TNF-α、IFN-γ及T-bet mRNA的相对表达量明显高于异体角膜移植+RvE1组和自体角膜移植组,差异均有统计学意义(均P＜0.05).结论 RvE1可抑制小鼠高危角膜移植排斥反应,作用机制可能与其下调角膜植片和淋巴细胞中Th1细胞及相关细胞因子的表达有关.     

深低温长期保存角膜穿透移植术后角膜内皮分析

目的 :了解深低温长期保存角膜移植术后植片内皮细胞的情况。方法 :应用深低温长期保存 3 0天～ 5 0个月 (平均 2 5 0± 11 9个月 )青壮年供体眼角膜完成穿透性角膜移植 2 4例 (2 4眼 )。供眼角膜深低温保存采用Kaufman Capella冷冻保存及复温技术。全部患者角膜植片一直保持透明 ,术后未曾出现移植免疫排斥反应 ,手术操作均由同一医生完成。术后 2～ 13 (平均 5 5± 2 2 )个月 ,应用接触式角膜内皮显微镜及与其同步显示的角膜内皮细胞分析仪对角膜植片中央进行观察和分析。结果 :角膜植片平均内皮细胞密度达 1769 6± 493 7个 /mm ,变异系数为 3 1 3± 11 4% ,六角形细胞出现率为 5 9 0± 6 9% ;植片内皮细胞密度与供体年龄、细胞面积变异系数呈负相关 ,与深低温保存时间以及受体年龄无关 ;六角形细胞出现率与植片内皮细胞密度呈正相关 ,与面积变异系数呈负相关 ,与术后随访时间无相关性。结论 :深低温长期保存角膜可满足穿透性角膜移植的需要。     

应用封闭式Dexsol基质保存角膜的研究

应用自制的Dexsol基质保存角膜，对40只动物(兔、猴各20只)角膜及5只人角膜进行了实验研究。保存时间为4℃者2周，34℃者5周。对保存在不同时间及不同温度的角膜应用锥蓝及茜素红双重染色，角膜内皮细胞活性良好。透射及扫描电镜观察保存2周(4℃)及4周(34℃)的角膜内皮细胞超微结构均无病理性改变。应用Dexsol基质保存的兔角膜进行同种穿透角膜移植，术后2个月，植片透明。20例接受Dexsol基质保存的供体角膜移植的患者，术后3个月移植片透明。植片中央部角膜内皮细胞密度平均为1915个／mm^2。     

小鼠胚胎干细胞条件培养基体外促进人角膜内皮细胞增殖的研究

目的　观察小鼠胚胎干细胞条件培养基（ｍｏｕｓｅｅｍｂｒｙｏｎｉｃｓｔｅｍｃｅｌｌｓｃｏｎｄｉｔｉｏｎｅｄｍｅｄｉｕｍ,ＥＳＣ-ＣＭ）是否可以在体外促进人角膜内皮细胞（ｈｕｍａｎｃｏｒｎｅａｌｅｎｄｏｔｈｅｌｉａｌｃｅｌｌｓ,ＨＣＥＣｓ）的增殖。方法　利用角膜内皮后弹力层组织块方法进行原代培养Ｐ０ＨＣＥＣｓ。实验组使用含有２５％ＥＳＣ-ＣＭ的培养液进行培养,对照组使用普通角膜内皮细胞培养液（ｃｏｒｎｅａｌｅｎｄｏｔｈｅｌｉｕｍｍｅｄｉ-ｕｍ,ＣＥＭ）进行培养。倒置相差显微镜、反转录聚合酶链反应（ｒｅｖｅｒｓｅ-ｔｒａｎｓｃｒｉｐｔｉｏｎｐｏｌｙｍｅｒａｓｅｃｈａｉｎｒｅａｃｔｉｏｎ,ＲＴ-ＰＣＲ）鉴定ＨＣＥＣｓ;倒置相差显微镜观察细胞的形态及萌出时间;ＷｅｓｔｅｒｎＢｌｏｔ、免疫组织化学法观察ＨＣＥＣｓ的泵相关功能蛋白（ｚｏｎａｏｃｃｌｕ-ｄｅｎｓｐｒｏｔｅｉｎ-１,ＺＯ-１）及Ｎａ＋-Ｋ＋-ＡＴＰ酶的表达。Ｇｉｅｍｓａ染色细胞克隆实验、免疫组织化学及流式细胞学检测Ｋｉ６７阳性率的方法比较ＨＣＥＣｓ的增殖能力;流式细胞学方法检查细胞周期及细胞凋亡情况。ＷｅｓｔｅｒｎＢｌｏｔ和免疫组织化学方法检测细胞周期负性调节蛋白Ｐ２１的水平,初步探讨其可能的作用机制。结果　原代培养时,２５％ＥＳＣ-ＣＭ组培养的ＨＣＥＣｓＰ２细胞爬出,细胞形态呈典型多角形结构。ＣＥＭ在Ｐ２时细胞形态变大,失去了多角形结构。２５％ＥＳＣ-ＣＭ 组和ＣＥＭ组均表达ＺＯ-１、Ｎａ＋-Ｋ＋-ＡＴＰ酶。２５％ＥＳＣ-ＣＭ组的Ｋｉ６７阳性率、克隆形成数量、进入到细胞周期Ｓ期和Ｇ２期的比例均高于ＣＥＭ组（均为Ｐ＜０．０５）。２５％ＥＳＣ-ＣＭ组的细胞凋亡数量和Ｐ２１阳性率均低于ＣＥＭ组（均为Ｐ＜０．０５）。结论　２５％ＥＳＣ-ＣＭ组可显著促进ＨＣＥＣｓ增殖;其作用可能是通过抑制Ｐ２１蛋白的表达和抑制细胞凋亡实现的,为ＨＣＥＣｓ体外大量扩增提供了一种新方法。     

脱细胞猪角膜基质体外支持角膜上皮和基质细胞的生长

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。     

Support of acellular porcine corneal stroma for growth of corneal epithelium and stromal cell in vitro

AIM: To determine whether acellular porcine cornea stroma (APCS) could support the growth of the rabbit corneal cells in vitro . METHODS: APCS was prepared. The rabbit's corneal epithelium and stromal cells were cultured and seeded on APCS in vitro . The observation of phase contrast photograph and histological examination were performed. RESULTS: Histological examination showed the epithelium grew on the scaffold of APCS in 2-3 layers at 10th day. The stromal cells adhered to the surface of the scaffold after 24 hours and invaded into the interlaminar of the material at 5th day. CONCLUSION: These results indicate that APCS can support the growth and proliferation of the corneal epithelium and stromal cells in vitro .     

Cultured human corneal endothelial cell transplantation with a collagen sheet in a rabbit model

PURPOSE: To evaluate the function of cultured human corneal endothelial cells (HCECs) in vivo and the feasibility of HCEC transplantation with a collagen sheet as the substitute carrier of HCECs. METHODS: Adult human donor cornea derived from cultured HCECs was labeled with the fluorescent tracker DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and seeded on a collagen sheet. The pump function of the HCEC sheet was evaluated by measurement of the potential difference and short-circuit current. A 6-mm sclerocorneal incision and Descemetorhexis were performed on rabbit eyes. The HCECs on a collagen sheet was brought into the anterior chamber and fixed to the posterior stroma (HCEC group). Rabbit corneas with collagen sheet transplantation after Descemetorhexis (collagen group) and with only Descemetorhexis (no-transplantation group) were the control. Each group, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations. RESULTS: Pump function parameters of the HCEC sheets were 76% to 95% of those of human donor corneas. Mean corneal thickness in the HCEC group was significantly less than in the collagen and no-transplantation groups 1, 3, 7, 14, 21, and 28 days (P < 0.05) after surgery. DiI-labeled cells were spread over the rear corneal surface in the HCEC group. Marked stromal edema was present in the collagen and no-transplantation groups with hematoxylin-eosin staining, but none in the HCEC group with collagen sheets bearing monolayer cells. CONCLUSIONS: The findings indicate that cultured HCECs transplanted from adult human donor cornea by means of a collagen sheet can retain their function of corneal dehydration in a rabbit model and suggest the feasibility of transplantation for CEC dysfunction using cultured HCECs with a collagen sheet.     

Cultivation of an immortalized human corneal endothelial cell population and two distinct clonal subpopulations on thermo-responsive carriers

Background  Recently, it was possible to show that human corneal endothelial cells (HCEC) can be cultured on thermo-responsive polymer substrates, and can be harvested as entire cell sheets without losing viability. We sought to study HCEC sheet cultivation on such cell culture carriers under serum-free conditions as the next consequential step in developing methods for generation of corneal endothelial cell transplants. Methods  An immortalized heterogenous HCEC population and two immortalized, clonally grown HCEC lines (HCEC-B4G12 and HCEC-H9C1) were cultured on thermo-responsive substrates under serum-supplemented and serum-free culture conditions. Cell sheets were characterized by phase contrast microscopy and by immunofluorescent staining for ZO-1, Na⁺,K⁺-ATPase, and vinculin. Results  All tested HCEC populations were able to adhere, spread and proliferate on thermo-responsive substrates under serum-supplemented conditions. Under serum-free conditions, pre-coating of the polymer substrates with ECM proteins was necessary to facilitate attachment and spreading of the cells, except in the case of HCEC-B4G12 cells. The heterogenous HCEC population formed closed monolayers, properly localized ZO-1 to lateral cell borders, and had moderate vinculin levels under serum-free, and higher vinculin levels under serum-supplemented culture conditions. HCEC-B4G12 cells formed closed monolayers, showed proper localization of ZO-1 and Na⁺,K⁺-ATPase to lateral cell borders, and had high vinculin levels irrespective of culture conditions. In contrast, HCEC-H9C1 cells had lowest vinculin levels under serum-supplemented, and higher vinculin levels under serum-free culture conditions. ZO-1 was detected throughout the cytoplasm under both culture conditions. These loosely adherent cells were only able to form a closed monolayer under serum-supplemented conditions. Conclusions  Serum-free production of HCEC sheets is possible. The extremely adherent clonal HCEC line B4G12 produced higher vinculin levels than the other two tested HCEC populations, and showed strong adherence to the thermo-responsive, polymeric culture substratum irrespective of culture conditions. This cell line closely resembles terminally differentiated HCEC in vivo, and was found to be particularly suitable for further studies on HCEC cell sheet engineering.     

体外培养的兔角膜内皮细胞生物学特性

背景如何选择高质量、生物学特性更接近在体生理状态的角膜内皮种子细胞是组织工程角膜研究的基础。角膜内皮细胞（CECs）的培养、鉴定及生物学特性检测是优选角膜内皮种子细胞的瓶颈问题。目的建立兔CECs原代培养及鉴定的方法,检测传代后细胞的生物学特性。方法从30只新西兰大白兔的角膜组织完整撕除后弹力层及CECs层,采用胰蛋白酶消化法进行原代培养,观察培养细胞的生长状态。分别从形态学、基因水平和蛋白水平鉴定细胞：茜素红染色法观察细胞形态,并与新鲜角膜组织的内皮细胞进行对比;逆转录聚合酶链反应（RT—PCR）法检测IVα2型胶原（COL4A2）、血管内皮生长因子受体2（FLK1）、Na^-K^＋ATP酶α亚单位（ATPIA1）、水通道蛋白1（AQP1）、电压依丛性阴离子通道（VDACs）等相对特异性基因;免疫细胞化学法观察神经元特异性烯醇化酶（NSE）、Na^-K^＋ATP酶及紧密连接蛋白ZO-1的表达。采用MTT比色法测定传代细胞的增生活性变化,采用超微量ATP酶试剂盒及免疫荧光法分别测定传代细胞Na^-K^＋ATP酶活性及其表达量的变化。结果原代培养的兔CECs多在24h内贴壁,2—3d达融合,形态呈六边形。随着传代代数的增加,细胞形态逐渐发生改变,第2代、第3代细胞可见空泡样变。原代培养的细胞茜素红染色可见清晰的细胞轮廓,与在体的CECs形态相似。RT—PCR法检测可见COL4A2、FLKl、ATP1Al、AQP1、VDACs等基因在培养细胞中表达。免疫荧光染色结果显示NSE、Na^-K^＋ATP酶、ZO-1在培养细胞中呈阳性表达。MTT检测结果显示,传代后细胞的增生活性逐渐下降,尤其第2代、第3代细胞下降明显。定量检测结果显示随着传代代数的增加,兔CECs的Na^-K^＋ATP酶活性逐渐降低,各代细胞的Na^-K^＋ATP酶活性总体比较的差异有统计学意义（F=77．174,P=0．000）。结论成功用酶消化法建立了体外培养兔CECs的方法,并从多个层次建立了纯化细胞的鉴定方法。研究结果证实经传代后的CECs的增生活性和功能均有所下降,因此在进行相关的研究时应选用前2代的细胞。     

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3 d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10 d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10 d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13 d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.     

A novel method of isolation, preservation, and expansion of human corneal endothelial cells

PURPOSE: To explore new strategies for effective isolation, preservation, and expansion of human corneal endothelial cells (HCECs). METHODS: Human corneal Descemet's membrane and corneal endothelial cells were digested with collagenase A or Dispase II in supplemented hormonal epithelial medium (SHEM) for 1.5 to 16 hours. HCEC aggregates derived from collagenase A digestion were preserved in serum-free medium with low or high calcium for up to 3 weeks. Cryosections of HCEC aggregates were subjected to immunostaining with ZO-1, connexin 43, type IV collagen, laminin-5, and perlecan, and apoptosis was determined by TUNEL or cell-viability assay. For expansion, HCEC aggregates were seeded directly or after brief treatment with trypsin/EDTA in SHEM, with or without additional bovine pituitary extract (BPE), nerve growth factor (NGF), or basic fibroblast growth factor (bFGF). The resultant HCECs were immunostained with ZO-1, connexin 43, and Ki67. RESULTS: Digestion with collagenase A, but not Dispase, of the stripped Descemet's membrane generated HCEC aggregates, which preserved cell-cell junctions and basement membrane components. High cell viability of HCEC aggregates was preservable in a serum-free, high-calcium, but not low-calcium, medium for at least 3 weeks. Brief treatment of HCEC aggregates with trypsin/EDTA resulted in a higher proliferation rate than without, when cultured in SHEM, and the resultant confluent monolayer of hexagonal cells retained cell-cell junctions. However, additional BPE, NGF, or bFGF did not increase cell proliferation, whereas additional BPE or bFGF disrupted cell-cell junctions. CONCLUSIONS: Collagenase A digestion successfully harvested aggregates with viable HCECs that were preservable for at least 3 weeks in a serum-free, high-calcium medium and, with brief trypsin/EDTA treatment, expanded in the SHEM into a monolayer with hexagonal cells that exhibited characteristic cell junctions.     

Cultured human corneal endothelial cell transplantation

Researchers have demonstrated the feasibility of transplanting human cultured corneal endothelial cells (HCEC) in various animal models. This review provides an overview of recent advances in our understanding of cultured corneal endothelial cell transplantation. We propose HCEC transplantation with a collagen sheet as the substitute carrier of HCEC. We also propose a novel strategy for corneal endothelial cell deficiency with the injection of adult human corneal endothelial precursors (HCEP). Using white rabbits or nude rats as keratopathy models, cultured HCEC were seeded on a collagen sheet. Descemetorhexis was performed on rabbit eyes. The HCEC collagen sheet was brought into the anterior chamber and fixed to the posterior stroma (HCEC group). Rabbit corneas with collagen sheet transplantation after descemetorhexis(collagen group) and with only descemetorhexis(no transplantation group) were the controls, respectively. As for HCEP transplantation, HCEP, isolated from rabbit corneal endothelial cells by sphere-forming assay, were injected into the anterior chamber and a face-down position was maintained for 24 hours in the rabbits (HCEP group). Pump function parameters of the HCEC sheets were 76-95% of those of human donor corneas. Mean corneal thickness in the HCEC group was significantly less than in the collagen and no transplantation groups 1, 3, 7, 14, 21, and 28 days (p< 0.05) after surgery. Cells were spread over the rear corneal surface in the HCEC group. In HE staining, marked stromal edema was present in the collagen and in the no transplantation groups, but not in the HCEC group with collagen sheets bearing monolayer cells. In the HCEP group, injected spheres were spread over the rear surface of the cornea and corneal edema was markedly suppressed. Our findings indicate that transplantation of cultured HCEC from adult human donor cornea by means of a collagen sheet can maintain the function of corneal dehydration. This suggests the feasibility of transplantation using cultured HCEC with a collagen sheet for corneal endothelial cell dysfunction. Additionally, adult precursor injection therapy can be also an effective strategy for corneal endothelial cell deficiency in place of conventional full-thickness corneal transplantation.