In vitro reconstruction and characterization of tissue-engineered human corneal epithelium with seeder cells from an untransfected human corneal epithelial cell line

AIM: To demonstrate the morphology and structure of in vitro reconstructed tissue-engineered human corneal epithelium (TE-HCEP) with seeder cells from an untransfected HCEP cell line. METHODS: The TE-HCEPs were reconstructed in vitro with seeder cells from an untransfected HCEP cell line, and scaffold carriers of denuded amniotic membrane (dAM) in air-liquid interface culture for 3, 5, 7 and 9 days, respectively. The specimens were examined with hematoxylin-eosin (HE) staining of paraffin-section, immunocytochemical staining, scanning and transmission electron microscopy. RESULTS: During in vitro reconstruction of TE-HCEP, HCEP cells formed a 3-4, 6-7 and 8-10 layers of an HCEP-like structure on dAMs in air-liquid interface culture for 3, 5 and 7 days, respectively. But the cells deceased to 5-6 layers and the structure of straified epithelium became loose at day 9. And the cells maintained positive expression of marker proteins (keratin 3 and keratin 12), cell-junction proteins (zonula occludens-1, E-cadherin, connexin 43 and integrin beta 1) and membrane transport protein of Na+-K+ ATPase. The HCEP cells in TE-HCEP were rich in microvilli on apical surface and established numerous cell-cell and cell-dAM junctions at day 5. CONCLUSION: The morphology and structure of the reconstructed TE-HCEP were similar to those of HCEP in vivo. The HCEP cells in the reconstructed TE-HCEP maintained the properties of HCEP cells, including abilities of forming intercellular and cell-extracellular matrix junctions and abilities of performing membrane transportation. The untransfected HCEP cells and dAMs could promisingly be used in reconstruction HCEP equivalent for clinical corneal epithelium transplantation.

Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models

AIM: To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS: TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS: After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575 mu m in thickness during the monitoring period. A 45 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION: The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.

人羊膜上皮干细胞对皮肤增生性瘢痕形成的作用

目的探讨人羊膜上皮细胞的干细胞对皮肤增生性瘢痕形成的作用。方法从足月分娩的人胎盘中剥离羊膜,采用胰蛋白酶消化法分离人羊膜上皮干细胞(human amniotic epithelial stem cells,hAECs),并在体外使用含表皮生长因子的LG-DMEM培养基进行培养,利用免疫荧光染色、流式细胞术和定向诱导等鉴定人羊膜上皮细胞的干细胞特性。构建兔耳全层皮肤缺损模型,左耳创面注射磷酸盐缓冲液(PBS)作为对照组,右耳创面注射羊膜上皮细胞悬液为实验组。结果实验中hAECs表达干细胞标志物SSEA-4和OCT-4,并表达间充质干细胞表面标记物,具有多向分化潜能。1个月后,兔子右耳瘢痕比左耳瘢痕薄,HE染色示:移植干细胞组有效的抑制了增生性瘢痕的形成,纤维化面积较小。在激光共聚焦细胞仪下可发现移植的hAESCs存活。结论人羊膜上皮细胞可以促进创面愈合,减少炎症反应,有效抑制兔耳创面瘢痕的形成。这可能成为瘢痕预防和治疗的新手段和切入点。

应用羊膜上皮干细胞微环境培养人角膜内皮细胞的研究

目的:建立一种利用羊膜上皮干细胞(human amniotic membrane epithelial cell,HAEC)微环境培养人角膜内皮细胞(human corneal endothelial cells,HCEC)的方法。方法:制备羊膜上皮干细胞微环境培养HCEC,并探讨诱导HCEC增殖的最佳培养微环境,倒置相差显微镜和透射电镜观察培养过程中细胞的形态学变化,MTT和Giemsa染色观察细胞增殖情况,Hoechst33342检测凋亡细胞比例。结果:在HCEC基本培养液(corneal endothelial cell medium,CEM)的基础上添加20%HAEC上清、HAEC-HCEC的微环境可促进HCEC的增殖,减少凋亡,细胞传代能力显著增强,HAEC-HCEC组传至4代仍保持多角形的内皮细胞形态。结论:羊膜上皮干细胞微环境培养可有效提高HCEE的增殖能力,更好地维持HCEC的形态,并能抑制其凋亡进程。

羊膜上皮细胞体外培养条件的优化及其干细胞标志的表达

本研究优化人足月胎盘羊膜上皮细胞(human amniotic epithelium cells,hAEC)的体外培养方法并观察hAEC的干细胞标志的表达情况。取健康产妇足月剖宫产术后的羊膜,采用胰酶多次消化获取hAEC,分别应用10%FBS的DMEM、类似胚胎干细胞的培养条件及添加表皮细胞生长因子(epidermal growth factor,EGF)的类似胚胎干细胞的培养条件对其进行原代和传代培养,观察培养后细胞形态,并通过流式细胞术检测、细胞免疫荧光染色等方法对培养细胞的干细胞多能性标志进行鉴定。结果表明:在类似胚胎干细胞培养条件的基础上添加10 ng/ml EGF可提高hAEC原代细胞的贴壁率和增殖能力,与广泛应用的10%FBS的DMEM培养条件相比,在此培养条件下细胞传代能力显著增强,传至5代仍保持上皮细胞表型,细胞形态在传代过程中改变不明显,并且表达胚胎干细胞全能性的表面标志SSEA-4,同时细胞免疫荧光染色显示培养后的hAEC表达波形蛋白(vimentin)。结论:采用类似胚胎干细胞的培养条件有利于hAEC在体外的扩增和传代,EGF对其增殖和传代有促进作用,培养的hAEC表达胚胎干细胞多能性标志。

人羊膜上皮细胞在妇产科相关疾病中的应用及研究进展

人羊膜上皮细胞(humanamniotic epithelial cells,hAECs)来源于羊膜的最内层,由胚泡内细胞团发育而来。hAECs可易分离,并具有部分胚胎干细胞(embryonic stem cells,ESCs)特性,在特定条件下,hAECs能分化为3个胚层的不同类型的细胞;hAECs还具有免疫原性低、免疫调节性和非致瘤性等优点。另外,hAECs可分泌多种细胞因子,如生长因子、神经营养因子和抗炎因子等。越来越多的研究表明,hAECs有助于修复患病受损的组织和器官并促进组织再生,因此具有广泛的应用前景。本文就hAECs在妇产科相关疾病(包括早发性卵巢功能不全、子宫内膜损伤、妇科恶性肿瘤、复发性流产)中的应用作一简要阐述。

人羊膜上皮细胞分化为结膜上皮细胞实验研究

目的探讨人羊膜上皮细胞在特定环境下诱导分化为结膜上皮细胞的可能性。方法人羊膜上皮细胞与去上皮结膜基质的结膜匀浆培养液孵育,角蛋白CK4、CK13、MUC5AC免疫组化鉴定细胞表型;过碘酸-Schiff试剂（PAS）;酶联免疫吸附（ELISA）法检测不同时间培养液内MUC5AC水平;逆转录-聚合酶链反应（RT-PCR）检测分化细胞MUC5AC基因表达。结果人羊膜上皮细胞经诱导培养后,免疫组化显示角蛋白CK4、CK13、MUC5AC表达阳性;PAS染色阳性;ELISA检测1~7 d内培养液中均有微量MUC5AC且逐渐升高;RT-PCR检测分化细胞MUC5AC表达阳性。结论人羊膜上皮细胞具有干细胞分化潜能,在特定条件下可定向分化为具有一定生理功能的结膜上皮细胞。

人羊膜上皮细胞在纤维蛋白支架上的生长情况及细胞生长因子对其生长的影响

目的人羊膜上皮细胞(human amniotie epithelial cells,HAEC)能否在体外构建的纤维蛋白支架上生长,以及表皮生长因子(epidema growth factor,EGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)和转化生长因子β1(transforming growth factor β1,TGF-β1)对HAEC增殖的影响。方法以纤维蛋白凝块为支架,构建HAEC生长模型,观察HAEC在纤维蛋白支架上生长情况(对照组),并加入细胞生长因子,包括EGF,bFGF,TGF-β1,观察细胞生长因子对HAEC生长的影响(研究组)。采用倒置显微镜、吉姆萨(Giemsa)染色和扫描电子显微镜观察HAEC的生长情况。采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)法分别检测纤维蛋白支架上HAEC凋亡情况。结果 HAEC在构建的纤维蛋白支架上生长良好。EGF组和bFGF组HAEC凋亡较对照组明显减少,差异有显著意义(P<0．05), TGF-β1组HAEC凋亡较对照组明显增加,差异有显著意义(P<0．05)。结论 HAEC能在构建的纤维蛋白支架上良好生长,EGF,bFGF可抑制HAEC凋亡,TGF-β1则促进HAEC凋亡。

人羊膜上皮细胞对急性肝衰竭动物模型的治疗效果

目的探讨人羊膜上皮细胞（hAECs）对急性肝衰竭（ALF）大鼠模型的治疗效果。方法从剖腹产人胎盘中分离羊膜，经过0．1％Trypsin-0．02％EDTA处理分离hAECs。hAECs用PKH26荧光生物素标记后经腹腔、门静脉、尾静脉三种不同途径注射到大鼠体内，观察细胞在肝脏分布情况，筛选出合适移植途径。选择240—260g雄性大鼠，采用D一氨基半乳糖（D—gal）2g/kg体重腹腔注射法建立ALF大鼠模型。模型制备1d后随机分为对照组和细胞治疗组。治疗组经腹腔直接注射hAECs，对照组注射等量的生理盐水。观察21d内大鼠存活率、肝功能和组织病理变化。结果免疫组化检测显示hAECs表达白蛋白、糖原等肝细胞标记。经门静脉、尾静脉途径移植细胞后可见肝脏内移植细胞定居，经腹腔途径移植的细胞在肝脏内未见分布，但可将多量细胞植入体内且操作简便。由于尾静脉途径移植细胞数量有限，门静脉途径具有创伤且注射后产生微循环栓塞等情况，本课题组选择腹腔直接注射途径实施细胞治疗。细胞移植后大鼠存活率提高，肝功能及细胞因子指标有明显改善，肝脏组织病理逐渐恢复正常。结论通过腹腔途径移植的hAECs对ALF大鼠模型有明显治疗作用。