人羊膜上皮细胞体外培养重建角膜上皮的实验研究

目的: 探讨人羊膜上皮细胞(human amniotic epithelial cells, HAECs)诱导分化为角膜上皮细胞的可塑性及其重建角膜上皮的可行性。方法: 取足月产人羊膜,通过胶原酶、胰蛋白酶和EDTA消化获得HAECs,将细胞在体外培养扩增、传代,将2~3代HAECs接种于去上皮的兔角膜基质上培养,待HAECs融合形成单层后,置于插入式培养皿中进行气液界面培养14 d。对角膜基质上培养的HAECs用光镜、扫描电镜和透射电镜进行形态学及超微结构观察,以及细胞角蛋白(CK)3/12的免疫组织化学检测。结果: HAECs可在角膜基质上生长,培养1~2 d可形成单层,气液界面培养14 d可形成4~5层细胞的复层上皮,扫描电镜观察细胞表面有丰富的微绒毛,透射电镜下可见上皮细胞之间有桥粒连接,免疫组化检测复层细胞表达CK3/12,所形成的复层结构与正常角膜上皮相似。结论: HAECs有可能作为种子细胞用于组织工程角膜上皮重建。     

兔角膜缘上皮干细胞体外扩增及生物学鉴定

背景:角膜缘干细胞体外培养的关键在于建立稳定的体外培养体系,包括角膜缘干细胞的定位、培养条件、载体选择和鉴别方法等.目的:探索兔角膜缘上皮干细胞体外扩增方法,并对其生物学特性进行鉴定.方法:采用兔角膜缘组织块培养法,以人羊膜为载体,在体外进行兔角膜缘上皮干细胞原代和传代培养.倒置显微镜观察其体外生长特征;苏木精-伊红染...     

羊膜接触镜输送人表皮干细胞重建兔角膜上皮

目的 利用眼表生物膜固定装置(BMFD)与羊膜(AM)制成接触镜,输送人表皮干细胞至角膜缘干细胞缺损(LSCD)的兔眼表,并评价其重建角膜上皮的效果.方法 制作去角膜上皮及角膜缘干细胞的雌性LSCD兔模型,分为3组:羊膜加人表皮干细胞移植组(n=20),将男性人表皮干细胞悬液注射到BMFD-AM接触镜(简称羊膜接触镜)与眼表之间;羊膜遮盖组(n=20),戴羊膜接触镜;对照组(n=20),单纯药物治疗.裂隙灯显微镜结合角膜荧光素钠染色观察并评价角膜修复情况.随访至角膜完全上皮化后,取角膜组织行病理学检查和免疫组织化学鉴定,PCR检测人Y-STR基因鉴定细胞来源.结果 羊膜加人表皮干细胞移植组角膜上皮修复快,平均(5.60±0.46)d达到完全上皮化,另外两组角膜上皮修复迟缓.羊膜遮盖组(9.25±0.51)d、对照组(12.45±0.65)d才完成角膜上皮化(P均<0.05).组织病理学示羊膜加人表皮干细胞移植组角膜上皮细胞形态接近正常,K3/K12(+)、Mucin 5AC(-)、K4(-).并可检测到人Y-STR基因.组织病理学示另外两组角膜上皮结膜化,K4(+)、Mucin 5AC(+)、K3/K12(-).结论 羊膜接触镜联合人表皮干细胞悬液注射能够重建LSCD兔角膜上皮.     

人羊膜上皮细胞的诱导分化

目的:建立体外培养及标记人羊膜上皮细胞(HAECs)的方法并应用原子力显微镜(AFM)从细胞形貌变化上探讨人羊膜上皮细胞向角膜上皮细胞转分化的可视性研究。方法:取足月产人羊膜,采用酶消化法获得HAECs进行原代和传代培养,对培养的细胞进行形态学观察和鉴定;HAECs与兔角膜基质细胞共培养2周,采用CK3+12免疫荧光细胞化学染色鉴定诱导后的细胞;应用AFM分别对共培养前后的人羊膜上皮细胞的表面超微结构进行观察,并与人角膜上皮细胞(HCECs)对比,分析细胞形貌的变化。结果:HAECs体外培养呈铺路石样外观,核/质比率小,连接成片。细胞形态为多角形,角蛋白keratin表达阳性,不表达CK3+12;免疫荧光显示共培养2周的HAECs表达CK3+12;AFM观察,共培养2周后HAECs细胞核中央由山谷样外观转变成类似HCECs的山峰样外观。结论:HAECs可成功进行原代和传代培养,在兔角膜基质细胞诱导培养条件下可向角膜上皮细胞转分化。     

体外培养人角膜缘上皮细胞抑制激活态角膜基质细胞的生长

背景：角膜受到损伤后,角膜基质细胞激活转变为成纤维细胞,引起角膜基质瘢痕化,导致视力下降甚至丧失。 目的：观察角膜不同部位上皮细胞与角膜基质细胞的相互作用,探索角膜缘上皮细胞群能否抑制激活态角膜基质细胞的生长。 方法：采用酶消化及机械外力相结合的方法获取人角膜中央、角膜旁中央及角膜缘处角膜上皮细胞与浅层角膜基质细胞,进行体外培养。相差显微镜下观察细胞形态及生长变化。待培养角膜上皮细胞与基质细胞发生接触抑制时,记作“0 周”,采用免疫荧光染色技术检测培养细胞中PCNA及p63蛋白的表达。 结果与结论：培养的角膜上皮细胞与成纤维细胞发生接触抑制时,两种细胞间有明显分界线。角膜缘组上皮细胞中PCNA及p63蛋白均有较高的表达;角膜旁中央组PCNA有较高的表达,p63蛋白阴性表达;角膜中央组PCNA表达较低,p63蛋白阴性表达;从鉴定结果中可以得出只有角膜缘组中存在一定比例的角膜缘上皮干细胞。角膜缘组上皮细胞逐渐包围并化解成纤维细胞,在相互作用4周后,成纤维细胞聚集成死细胞团,缺乏角膜缘干细胞的中央组及旁中央组中成纤维细胞生长面积增加,上皮细胞生长受到抑制甚至死亡。说明体外培养的角膜缘上皮细胞群可以抑制激活态角膜基质细胞的生长。     

改良组织块培养法体外培养人角膜上皮干细胞

文题释义：角膜上皮干细胞：属于单能干细胞,具有细胞周期长、低分化状态、增殖潜力大、不对称分裂等特点,定位于角膜缘基底细胞层,又称之为角膜缘干细胞,对角膜上皮细胞更新及维持角膜透明起着重要作用。角膜缘干细胞的体外培养方法：主要包括酶消化培养法和组织块培养法。酶消化培养法是利用DispaseⅡ酶破坏角膜缘上皮细胞与基底膜之间的半桥粒连接,然后剥取角膜缘上皮层,再使用胰酶将其消化为单个细胞进行培养。组织块培养法没有经过酶的双重消化,将剖取的角膜缘组织块进行贴壁,细胞游离出组织块进行贴壁生长,需要一个漫长的过程。  摘要背景：角膜上皮干细胞定位于角膜缘,又称之为角膜缘干细胞,临床上由于眼表严重热烧伤、化学性烧伤、慢性炎症等原因引起的角膜缘干细胞缺乏或功能障碍治疗较为棘手。目前利用组织工程技术体外培养角膜上皮干细胞并进行临床移植成为新型有效的治疗方向。目的：探讨在无血清培养条件下采用改良组织块培养法培养人角膜上皮干细胞的可行性。方法：人角膜缘组织来自河南省眼库,植片直径小于8 mm角膜移植术后的供者剩余眼球材料,手术显微镜下剖取角膜缘上皮层外2/3区域,采用2种方法培养人角膜上皮干细胞,常规组织块培养组是将组织块上皮面向上贴壁,加入K-SFM培养液后置于 37 ℃、体积分数为5%CO₂细胞培养箱中培养;改良组织块培养组是先将组织块浸泡于K-SFM培养液中,置于细胞培养箱中孵育12 h,然后组织块上皮面向下贴壁培养。组织块周边有细胞游离出贴壁生长记作“培养第1天”,每日相差显微镜下观察细胞生长变化。利用免疫荧光染色技术检测改良组织块培养第5,10,14天时原代细胞中p63及K3的表达。结果与结论：①改良组织块培养组出膜时间明显短于常规组织块培养组(P < 0.05),出膜率明显高于常规组织块培养组(P < 0.05);②改良组织块培养组细胞生长状态良好,培养第10天可见小体积细胞较多,聚集成灶状分布;培养第14天可见细胞克隆灶,克隆灶内细胞体积较小,形态均一;③培养第5天,K3表达量较多,p63表达量较少;培养第10天,K3和p63表达量均增多;培养第14天,K3表达量未见明显增多,p63表达量明显增多;④在无血清培养条件下,改良组织块培养法能显著促进角膜上皮干细胞的游离,提高体外培养细胞数量,为人角膜缘上皮组织片的构建提供种子细胞。ORCID: 0000-0001-8370-174X(许中中) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

“去细胞”羊膜基质种植人脐静脉内皮细胞的实验研究

目的制备“去细胞”羊膜基质(HAAM),观察内皮细胞在HAAM上的种植效果及细胞种植后的一氧化氮(NO)分泌功能和粘附能力,为进一步研究以HAAM覆盖修饰血管内支架并种植内皮细胞治疗再狭窄提供理论和实验依据。方法胰酶消化去除羊膜上皮细胞,保留基底膜作为载体,种植人脐静脉内皮细胞(HUVEC),体外培养形成单层,并进行细胞形态观察、NO分泌功能及细胞粘附力测定。结果HUVEC可在HAAM上粘附生长并增殖,体外培养67天融合形成单层,其形态、分泌NO功能及细胞粘附力均优于对照组培养的内皮细胞。结论羊膜基质可作为内皮细胞生长粘附的载体并增强其功能。     

PCNA在新西兰大耳兔角膜及角膜缘的表达

目的 观察增殖细胞核抗原(PCNA)在新西兰大耳兔角膜上皮的表达,进一步了解角膜缘干细胞(LSCs)的增殖情况.方法 健康成年新西兰大耳兔30只,体重2.0～2.5 kg,雌雄错配,取双侧眼球,角膜上、下、内、外四个部位常规石蜡切片,采用常规组织学HE染色、免疫组织化学SABC法染色及图像分析进行研究,相关数据做统计学处理.结果 HE染色显示角膜缘上皮细胞排列紧密而不规则,层数较角膜中央部多,基底层细胞呈矮柱状或立方形,核大而圆,居中着色较深,基底部可见明显乳头样突起.角膜中央上皮基底层细胞较大,呈柱状.免疫组织化学SABC法显示PCNA阳性细胞,核卵圆而大,多位于角膜上、下方角膜缘上皮基底层,在角膜缘浅层及角膜浅层有少量分布,在角膜中央上皮基底层可见极少PCNA阳性细胞出现.图像分析显示每个高倍镜视野中上、下角膜缘与角膜及内、外角膜缘PCNA阳性细胞数量及灰度具有显著性差异(P＜0.05).结论 PCNA在角膜上皮的标记细胞主要是LSCs和短暂扩充细胞,即标记处于迅速增殖中的细胞.PCNA在角膜中央及角膜缘的表达反映了LSCs增殖,分化的过程;同时提示上、下方的角膜缘干细胞分布较多.     

体外诱导人羊膜上皮细胞分化为结膜上皮样细胞

目的将人羊膜上皮细胞(HAEC)和兔结膜上皮细胞共培养,观察是否能将HAEC诱导分化为结膜样上皮细胞。方法分别采用酶消化法和组织块法获得HAEC和兔结膜上皮细胞,并对培养的细胞进行形态学观察;运用TranswellTM非接触共培养系统将HAEC和兔结膜上皮细胞共培养2周,诱导HAEC分化;通过免疫荧光组织化学染色法检测诱导后HAEC中细胞角蛋白4(CK4)、黏蛋白Muc4、Muc5AC的表达,实时定量PCR(qRT-PCR)检测分化细胞Muc4、Muc5AC mRNA的表达。结果体外培养的人羊膜上皮细胞和兔结膜上皮细胞形态较为相似,免疫荧光组织化学染色法检测到诱导后HAEC中CK4、Muc4、Muc5AC呈阳性表达,qRT-PCR检测分化细胞Muc4、Muc5AC mRNA表达阳性。结论 HAEC在与兔结膜上皮细胞共培养的诱导条件下,可以向结膜上皮样细胞和产黏蛋白细胞转分化。     

诱导骨髓间质干细胞分化为角膜上皮样细胞的初步研究

目的: 观察人骨髓间质干细胞（MSCs）移植到兔角膜基质后的分化发育情况，探讨MSCs分化为角膜上皮细胞的可行性。方法: 24只新西兰兔随机分为2组。实验组：将人MSCs接种在保存人羊膜上培养4 d，用5-溴脱氧尿嘧啶（BrdU）标记后移植到兔角膜基质；对照组：采用保存羊膜移植到兔角膜基质。分别于移植后1、2、3、4、6和8周，摘取各组实验眼行组织学和免疫组织化学检查，检查移植到兔角膜基质的MSCs的存活、形态变化以及移植局部的反应等情况；免疫组织化学检测移植到角膜基质的带有BrdU标记的细胞角蛋白K3/12（CK3/CK12）和角蛋白K13（CK13）的表达。结果: MSCs接种到羊膜后能在羊膜上生长，与羊膜共培养4 d后，MSCs贴附羊膜生长迅速，组织学特征无明显改变。羊膜负载MSCs移植到兔角膜基质表面, 术后免疫组织化学检测角膜上皮层CK3/CK12表达阳性， CK13表达阴性，在重建的角膜上皮层可检测到BrdU核阳性细胞并同时表达角膜上皮细胞特异性表面标志蛋白K3/K12，未发生免疫排斥反应，未见异常增殖细胞。结论: 羊膜负载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。     

The phenotype of bovine corneal epithelial cells on chitosan membrane

Corneal wound healing is one of the major issues in ocular surface reconstruction and ocular surface diseases. Amniotic membrane (AM) transplantation is an excellent treatment modality to promote corneal wound healing and treat corneal diseases. It is interesting and valuable to search for another synthetic and biocompatible substitute for the study of mechanism of AM and the treatment of ocular surface disorders. Chitosan, the second-most abundant polymer in nature, has many biological advantages such as biocompatibility, biodegradability, hemostatic activity, and wound-healing property to be used as biomedical applications. The purpose of this project is to evaluate the phenotype of cultured corneal epithelial cells in vitro on synthetic chitosan membrane (CM). We cultivated bovine corneal epithelial cells on CM and AM, and then evaluated their phenotypes. The viability of the respective cell cultures was investigated using the 3-[4,5-dimethylrhiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The cytotoxicity of CM and AM to corneal epithelial cells was evaluated by lactate dehydrogenase (LDH) assay. The morphology of cultivated corneal epithelial cells on CM and AM was observed by scanning electron microscopy. Additionally, immunocytochemical stainings were used to confirm the phenotype of corneal epithelial cells. In MTT and LDH assays we found that the CM can support the growth of cultured corneal epithelial cells in good condition with minimal toxicity. The SEM and immunohistocytochemistry showed that the phenotype of corneal epithelial cells is compatible with that of AM. We conclude that the CM has the potential to be a suitable biomaterial for treating ocular surface disorders.     

The effect of in vivo grown corneal epithelium transplantation on persistent epithelial defects with limbal stem cell deficiency

We report our experience with corneal epithelium, grown in vivo, transplantation in three patients with persistent epithelial defect (PED). The three patients had ocular surface disease unresponsive to standard treatments and were therefore chosen for transplantation. They underwent transplantation of epithelial sheets, grown in vivo, to the most affected eye. In vivo cultivation was carried out in the cornea of a living related donor. After epithelialization was completed, the epithelium grown on an amniotic membrane was harvested gently; it was then transplanted into the patient's eye after debridement of fibrovascular tissue. The cultivated epithelium was completely epithelialized by 2 weeks; it was well-differentiated with well-formed hemidesmosome. On immunohistochemical staining, p63, connexin 43, and Integrin beta4 were expressed in the cells on the epithelial sheet. The PED was covered completely and maintained for 4 weeks in all cases. However, corneal erosion recurred after 5 weeks in two cases. This novel technique demonstrates the corneal epithelial cells can be expanded in vivo successfully on denuded amniotic membrane of a healthy cornea and harvested safely. A corneal epithelial sheet, grown in vivo, can be transplanted to treat eye with a severe ocular surface disease, such as total limbal deficiency.     

Ultrastructural analysis of in vivo expanded corneal epithelium on amniotic membrane

The purpose of this study is to characterize and compare the ultrastructural changes occurring during the in vivo cultivation of corneal epithelium on amniotic membrane (AM) at several different time points. Corneal burn patients (n=7) with a corneal epithelial defect and severe limbal damage were selected. Initially, AM transplantation with limbal autograft was performed at the acute stage of corneal burn to reconstruct the damaged ocular surface. One to six (mean interval; 3.3+/-1.2) months later, the central part of AM containing an in vivo expanded corneal epithelium was excised and retransplanted in adjacent lesions. The excised epithelium with AM was examined by electron microscopy and immunohistochemical study. By electron microscopy, one and two months after expansion, cultivated epithelium on AM showed an undifferentiated epithelium and an incomplete basement membrane (BM). But, after three months, the cultivated epithelium began to differentiate into a multilayered epithelium with a continuous BM with increased hemidesmosomes. These findings were further confirmed by immunohistochemical study, that cytokeratin K3 was expressed in the cultivated corneal epithelium and newly formed BM was partially positive of collagen IV at three months. At least 3 months may be needed for the proliferation and differentiation of in vivo cultivated corneal epithelium on AM.     

Amniotic membrane immobilized poly(vinyl alcohol) hybrid polymer as an artificial cornea scaffold that supports a stratified and differentiated corneal epithelium

Poly(vinyl alcohol) (PVA) is a biocompatible, transparent hydrogel with physical strength that makes it promising as a material for an artificial cornea. In our previous study, type I collagen was immobilized onto PVA (PVA-COL) as a possible artificial cornea scaffold that can sustain a functional corneal epithelium. The cellular adhesiveness of PVA in vitro was improved by collagen immobilization; however, stable epithelialization was not achieved in vivo. To improve epithelialization in vivo, we created an amniotic membrane (AM)-immobilized polyvinyl alcohol hydrogel (PVA-AM) for use as an artificial cornea material. AM was attached to PVA-COL using a tissue adhesive consisting of collagen and citric acid derivative (CAD) as a crosslinker. Rabbit corneal epithelial cells were air-lift cultured with 3T3 feeder fibroblasts to form a stratified epithelial layer on PVA-AM. The rabbit corneal epithelial cells formed 3-5 layers of keratin-3-positive epithelium on PVA-AM. Occludin-positive cells were observed lining the superficial epithelium, the gap-junctional protein connexin43-positive cells was localized to the cell membrane of the basal epithelium, while both collagen IV were observed in the basement membrane. Epithelialization over implanted PVA-AM was complete within 2 weeks, with little inflammation or opacification of the hydrogel. Corneal epithelialization on PVA-AM in rabbit corneas improved over PVA-COL, suggesting the possibility of using PVA-AM as a biocompatible hybrid material for keratoprosthesis.     

The impact of compositional topography of amniotic membrane scaffold on tissue morphogenesis of salivary gland

Amniotic membrane (AM) has been widely used in the reconstruction of oral epithelial defects. However, whether it is also effective in facilitating tissue formation of salivary gland, an appendix of oral epithelia, has never been explored. To investigate the effects and the underlying mechanism of AM on salivary gland morphogenesis, murine fetal submandibular gland (SMG) explants were cultured on different preparations of AM scaffolds. It was found that, on AM stromal scaffold, SMG demonstrated well-developed branching morphogenesis. Nonetheless, on AM epithelial scaffold, SMG epithelial cell converted to a spindle-shape, lost intercellular connection, changed cytoskeletal organization, and exhibited scattering behaviors. Meanwhile, the integrity of SMG basement membrane was dismantled as well. However, when acellular AM epithelial scaffold was used, cultured SMG demonstrated organized morphology, indicating that AM epithelial component provided specific surface features for SMG morphogenesis. To further investigate AM scaffold morphogenetic effect, it was found hepatocyte growth factor (HGF), an epithelial scattering factor, was expressed abundantly in cultivated AM epithelia. After blocking HGF function of AM, cultured SMG regained branching activity, reorganized cell adhesion and subcellular organization, and reproduced basement membranes. Therefore, AM-derived bioactive factor profoundly influences cell behaviors and structure formation of SMG. Together, this study showed that compositional topography of AM scaffold is important in affecting SMG morphogenesis. By understanding the effects of AM scaffold on SMG morphogenesis, it provides important information for rationally designing and fabricating AM scaffold for salivary gland regeneration.     

Cytocompatibility of Three Corneal Cell Types with Amniotic Membrane

Rabbit limbal corneal epithelial cells, corneal endothelial cells and keratocytes were cultured on amniotic membrane. Phase contrast microscope examination was performed daily. Histological and scan electron microscopic examinations were carried out to observe the growth, arrangement and adhesion of cultivated cells. Results showed that three corneal cell types seeded on amniotic membrane grew well and had normal cell morphology. Cultured cells attached firmly on the surface of amniotic membrane. Corneal epithelial cells showed singular layer or stratification. Cell boundaries were formed and tightly opposed. Corneal endothelial cells showed cobblestone or polygonal morphologic characteristics that appeared uniform in size. The cellular arrangement was compact. Keratocytes elongated and showed triangle or dendritic morphology with many intercellular joints which could form networks. In conclusion, amniotic membrane has good scaffold property, diffusion effect and compatibility with corneal cells. The basement membrane side of amniotic membrane facilitated the growth of corneal epithelial cells and endothelial cells and cell junctions were tightly developed. The spongy layer of amniotic membrane facilitated the growth of keratocytes and intercellular joints were rich. Amniotic membrane is an ideal biomaterial for layering tissue engineered cornea.     

纤维蛋白胶为载体构建人羊膜上皮细胞植片的实验研究

目的： 探讨利用纤维蛋白胶作为支架构建组织工程人羊膜上皮细胞(HAECs)植片重建眼表的可行性。方法：取足月剖宫产胎盘羊膜,经胶原酶和胰蛋白酶消化后,获得HAECs。在体外构建的纤维蛋白胶片上培养HAECs，细胞融合成片后，利用气液界面复层化，采用倒置显微镜、组织切片、HE 染色、细胞角蛋白免疫组织化学染色和扫描电子显微镜观察HAECs的生长情况。结果：HAECs在纤维蛋白胶表面生长良好，细胞呈圆形或多角形,长满后呈上皮细胞特有的铺路石样外观，扫描电镜观察细胞表面有丰富的微绒毛，细胞广谱角蛋白单克隆抗体染色阳性。细胞有复层生长趋势，植片较为透明。结论：以纤维蛋白胶为载体构建组织工程人 HAECs 植片，具有眼表重建的潜在运用价值。     

Reconstructed human cornea produced in vitro by tissue engineering.

The aim of the present study was to produce a reconstructed human cornea in vitro by tissue engineering and to characterize the expression of integrins and basement membrane proteins in this reconstructed cornea. Epithelial cells and fibroblasts were isolated from human corneas (limbus or centre) and cultured on plastic substrates in vitro. Reconstructed human corneas were obtained by culturing epithelial cells on collagen gels containing fibroblasts. Histological (Masson's trichrome staining) and immunohistological (laminin, type VII collagen, fibronectin as well as beta1, alpha3, alpha4, alpha5, and alpha6 integrin subunits) studies were performed. Human corneal epithelial cells from the limbus yielded colonies of small fast-growing cells when cultured on plastic substrates. They could be subcultured for several passages in contrast to central corneal cells. In reconstructed cornea, the epithelium had 4-5 cell layers by the third day of culture; basal cells were cuboidal. The basement membrane components were already detected after 3 days of culture. Integrin stainings, except for the alpha4 integrin, were also positive after 3 days. They were mostly detected at the epithelium-stroma junction. Such in vitro tissue-engineered human cornea, which shows appropriate histology and expression of basement membrane components and integrins, provides tools for further physiological, toxicological and pharmacological studies as well as being an attractive model for gene expression studies.