构建组织工程化羊膜角膜上皮植片的研究进展

虽然组织工程化角膜组织已成功构建,并为体外进行角膜生理、病理、药理和角膜移植等方面的研究提供了与活体角膜很接近的模型,但其距离临床应用还有一定距离。近年来,采用组织工程技术构建的羊膜角膜上皮植片已率先应用于临床,为成千上万的由于角膜上皮缺损所致的角膜疾病患者带来希望,本文对有关羊膜的基础研究、羊膜负载角膜缘干细胞植片的构建方法及其功能的评价、角膜缘干细胞在羊膜上的生物学特性、植片移植及术后护理等方面的研究进展和存在的问题进行综述。     

小鼠角膜上皮细胞消化培养法和组织块培养法的比较研究

目的:比较小鼠角膜上皮细胞消化培养法和组织块培养法。方法:分别使用消化培养法和组织块培养法培养小鼠角膜上皮细胞。比较两种方法中小鼠角膜上皮细胞的克隆形成率(CFE)和群体倍增(PD)。通过Western blotting方法检测p63、角蛋白19以及角蛋白12的表达。结果:其中80%组织块培养法的原代培养可成功传代,而仅12%的消化培养法原代培养可成功传代;两者比较有显著性差异(P<0.05)。传代培养中,组织块培养法中55%的第一代(P1)细胞可以传代超过P10并继续稳定传代至少可传至P25。而消化培养法传代至P2即不能融合。在P1,组织块培养法细胞的CFE高于消化培养法(P=0·02);而组织块培养法P20细胞的CFE又显著高于其P1细胞(P=0.001)。免疫荧光染色显示消化培养法的P1细胞和组织块培养法的P1,P20细胞均表达p63和K19。K12仅在消化培养法的P1细胞和组织块培养法的P1中表达,而组织块培养法的P20细胞中,K12阴性表达。结论:小鼠角膜上皮细胞的培养,组织块培养法优于消化培养法。     

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.     

组织工程构建角膜上皮中相关种子细胞的研究进展

传统角膜移植术存在着供体来源缺乏、术后免疫排斥等问题.虽然很多地方已建立了眼库,但供体来源依然有限.因此,构造一种类似正常角膜结构的组织工程角膜、扩大角膜移植供体来源、满足患者需求成为治疗严重眼表疾病需要迫切解决的问题.本文对角膜上皮细胞的组织工程构建中采用的种子细胞进行归类,并介绍相关的重建方法及分析各种种子细胞在角膜上皮细胞组织工程中的优势及存在的问题,以期能为该领域的研究提供有益的探索方向.     

兔舌黏膜细胞构建组织工程化角膜上皮的实验研究

目的探讨兔舌黏膜上皮细胞以去上皮羊膜为载体,体外构建组织工程角膜上皮的可行性和方法,试图为眼表重建寻求另一种来源丰富的有效组织工程化角膜移植材料。方法胰酶消化去除羊膜上皮细胞,保留基底膜作为载体,将兔舌黏膜上皮细胞种植于去上皮羊膜基底膜进行体外培养扩增。对培养获得的复合上皮片进行形态观察、常规病理切片HE染色和透射电镜观察。采用AE1/AE3单克隆抗体鉴定复合上皮片中有无角蛋白表达。结果兔舌黏膜上皮细胞在去上皮羊膜基底膜面能黏附生长并增殖,体外培养2～3周左右即可在羊膜上汇合成片并分层。形成的复合上皮片由羊膜基底膜和2～3层较扁平的上皮细胞组成,细胞之间可见桥粒连接,细胞表达角蛋白。结论兔舌黏膜上皮细胞以去上皮羊膜为载体,经体外培养扩增获得的复层组织,初具角膜上皮的雏形结构。     

组织工程角膜上皮治疗兔角膜缘干细胞缺乏症的研究

目的 评价以纤维凝胶膜为载体,体外培养自体角膜缘干细胞移植术治疗角膜缘干细胞缺乏症的临床效果。方法 制作兔眼角膜缘干细胞完全缺乏模型,取对侧眼角膜缘组织进行干细胞培养,并以纤维凝胶膜为载体制成组织工程角膜上皮。将实验动物随机分为4组,其中Ⅰ-Ⅲ组为实验组,行组织工程角膜上皮移植术,Ⅰ组移植术后观察3个月,Ⅱ组移植术后观察1个月,Ⅲ组移植术后观察2周。Ⅳ组为对照组,移植不含干细胞的凝胶膜,术后观察3个月。以角膜上皮染色、角膜混浊及新生血管3项指标进行临床疗效评定,通过病理检查评估术后不同时期角膜上皮修复情况,印迹细胞学检查移植前后角膜上皮的细胞表型,免疫组织化学染色观察角膜上皮特异性角蛋白K3、MUCSAC及转录因子p63在移植角膜上皮的表达。结果 实验组角膜上皮逐渐愈合,透明度提高,新生血管减退或消失;对照组角膜呈持续混浊,上皮缺损迁延不愈,最终大量新生血管长入,上皮结膜化。临床指标评分比较,差异有统计学意义（P=0．021）。角膜上皮印迹细胞学检查显示对照组为PAS（＋）的结膜细胞表型,而实验组上皮细胞PAS（-）。组织病理学显示实验组为正常角膜上皮结构,对照组为结膜化生的上皮,可见血管和杯状细胞。免疫组织化学显示实验组角膜上皮表达角膜上皮特异性角蛋白l（3（AE5）,不表达结膜特异性MUCSAC,在上皮基底部表达转录因子p63。对照组上皮持续表达MUCSAC。结论 组织工程角膜上皮移植术是一种有效的治疗角膜缘干细胞缺乏症的方法,可重建眼表,修复角膜上皮的损伤,抑制角膜上皮结膜化和新生血管。纤维凝胶膜为一种新型的组织工程材料,吸收快,透明度高,具有良好的组织相容性,是一种较理想的移植载体材料。（中华眼科杂志．2006．42：679-685）     

组织工程角膜上皮移植治疗兔角膜碱烧伤的形态学观察

目的:探讨组织工程角膜上皮移植治疗角膜碱烧伤的疗效和时机。方法:1mol/LNaOH制作改良兔角膜碱烧伤模型21只42眼,分对照组和移植组,移植组分别在碱烧伤后1,3,6,9d(早)和14d(中)行自体或同种异体组织工程角膜上皮移植术,比较移植组及对照组烧伤后28d内眼表和组织病理学变化。结果:角膜碱烧伤后7d开始出现角膜上皮的大片脱落,14d角膜上皮大片脱落或溃疡发生率达72%,持续至28d,而移植组在28d时发生率仅为25%,大多获得完整的角膜上皮;烧伤后早期移植组角膜基质深层炎性细胞浸润和新生血管生长较对照组明显受到抑制,而中期移植组角膜基质层较对照组并无明显差异;28d内异体组织工程角膜上皮移植的免疫排斥反应并不大于自体移植。结论:自体或同种异体组织工程角膜上皮移植均可尽快恢复眼表完整性,且烧伤后早期移植效果明显优于中期移植。     

组织工程角膜上皮支架材料研究进展

体外培养角膜缘干细胞构建组织工程角膜上皮后再行角膜移植是治疗角膜缘干细胞缺乏导致眼表疾病的重要方法,选择理想的支架材料是构建组织工程角膜上皮过程中的一个关键环节。但支架材料的组织相容性、透光度,支架材料的降解与角膜修复的同步化,手术的长期有效性等问题仍有待进一步研究。细胞生物学、分子生物学、组织工程学和材料学的发展为构建组织工程角膜上皮的研究开辟了更广阔的前景。从近年来新发现或研制的支架材料的来源、优缺点及应用等方面,就构建组织工程角膜上皮支架材料的研究进展进行综述。     

角膜上皮细胞培养的研究

角膜上皮细胞的重要性已逐渐为人们所重视,对角膜上皮细胞的基础和临床研究亦日渐深入。角膜上皮细胞的组织培养作为研究角膜上皮细胞的一种很好的技术方法,已被很多实验室广泛应用于角膜上皮细胞的胶原、胶原酶代谢产物,粘蛋白代谢产物,病毒感染的影响,细胞分化,各种生长促进因子和药物对上皮细胞生长的影响,基底膜胶原     

组织工程化角膜研究进展

组织工程(tissue engineering,TE)是一门跨越医学生物学、工程设计学、材料科学等的交叉科学,近20a来,包括种子细胞、支架材料、组织构建等都取得了长足进步。TE化角膜对于角膜疾病是一种很有希望的治疗方法,因而我们对近年来TE角膜的研究做一回顾,以探索该领域存在的问题和进一步的研究发展方向。     

Comparative study of four culture methods to engineer murine corneal epithelial sheet

AIM: To investigate the roles of feeder cells in stratification of murine corneal epithelial cells and build an ideal method to engineer stratified epithelial sheet. METHODS: Using contact feeder culture, separated feeder culture, compound feeder culture and culture without feeder cells by air-lifting method in Transwell chamber culture system, tissue engineered corneal epithelium was reconstructed. Corneal sheets were stained with hematoxylin and eosin (HE) for histological observation. The expression of p63 and keratin 19 (K19) and involucrin (IVL) was investigated by immunocyto- chemistry analysis. RESULTS: Stratification was limited to three to four layers in the contact feeder group, whereas separate feeder sheets were slightly more stratified. The compound feeder group produced a stratified epithelium with five to seven layers of cells. The group without 3T3 feeder cells formed only two to three layers of cells. Immunostaining images in the compound feeder group showed expression of progenitor markers p63 and K19 in the basal and suprabasal layer, as well as differentiation marker involucrin in all layers. CONCLUSION: The remarkable stratification as well as the limbal phenotype makes the compound feeder system a candidate tool for cultivating transplantable epithelial sheets.     

Effect of calcium on the proliferation and differen-tiation of murine corneal epithelial cells in vitro

AIM: To investigate the effect of calcium on the proliferation and differentiation of murine corneal epithelial cells in vitro. METHODS: Mouse corneal epithelial cells were cultured in serum-free low-Ca2+ medium (KSFM) and KSFM supplemented with 0.9mmol/L Ca2+. Population doublings (PDs) were determined. The expression of corneal epithelial cell markers p63, keratin 19 (K19) and involucrin was investigated by RT-PCR analysis and semiquantitative analysis of Western blotting. RESULTS: Cells in KSFM were stably subcultured over 25 passages, however, none of the cell lines could pass P4 in KSFM with Ca2+. In KSFM, the cells was were homogeneous and small cells with typical cobblestone appearance; and expressed p63, K19 and involucrin. After medium was supplemented with calcium, cells became a heterogeneous mix of small and large cells. Furthermore, semiquantitative analysis of Western blotting showed that the expression of involucrin was increased significantly. CONCLUSION: Calcium has the effect of inhibiting pro- liferation and triggering differentiation on mouse corneal epithelial cells.     

Effect of calcium on the proliferation and differen-tiation of murine corneal epithelial cells in vitro

AIM: To investigate the effect of calcium on the proliferation and differentiation of murine corneal epithelial cells in vitro.METHODS: Mouse corneal epithelial cells were cultured in serum-free low-Ca2+ medium (KSFM) and KSFM supplemented with 0.9mmol/L Ca2+.Population doublings (PDs) were determined.The expression of corneal epithelial cell markers p63,keratin 19 (K19) and involucrin was investigated by RT-PCR analysis and semiquantitative analysis of Western blotting.RESULTS: Cells in KSFM were stably subcultured over 25 passages,however,none of the cell lines could pass P4 in KSFM with Ca2+.In KSFM,the cells was were homogeneous and small cells with typical cobblestone appearance;and expressed p63,K19 and involucrin.After medium was supplemented with calcium,cells became a heterogeneous mix of small and large cells.Furthermore,semiquantitative analysis of Western blotting showed that the expression of involucrin was increased significantly.CONCLUSION: Calcium has the effect of inhibiting proliferation and triggering differentiation on mouse corneal epithelial cells.     

胎牛血清对小鼠角膜上皮细胞生长和分化的影响(英文)

目的:探讨胎牛血清对小鼠角膜上皮细胞生长和分化的影响。方法:分别在无血清低钙培养基(KSFM)和含100mL/L胎牛血清的KSFM中培养小鼠角膜上皮细胞。比较两种培养基中角膜上皮细胞的群体倍增(PD)。通过RT-PCR和Western blotting方法检测p63、角蛋白19以及involucrin的表达。结果:在KSFM中,小鼠角膜上皮细胞可稳定传代超过25代,但在含胎牛血清的培养基中,细胞仅能存活3代。在KSFM中,培养细胞呈典型铺路石样外观,RT-PCR和Western blotting结果显示细胞表达p63、角蛋白19以及in-volucrin。当培养基中加入胎牛血清后,细胞形态明显增大,呈扁平状; involucrin表达显著增强。结论:胎牛血清可抑制小鼠角膜上皮细胞增生、诱导其分化。     

胎牛血清对小鼠角膜上皮细胞生长和分化的影响

目的：探讨胎牛血清对小鼠角膜上皮细胞生长和分化的影响。方法：分别在无血清低钙培养基（KSFM）和含100mL／L胎牛血清的KSFM中培养小鼠角膜上皮细胞。比较两种培养基中角膜上皮细胞的群体倍增（PD）。通过RT-PCR和Westernblotting方法检测p63、角蛋白19以及involucrin的表达。结果：在KSFM中,小鼠角膜上皮细胞可稳定传代超过25代,但在含胎牛血清的培养基中,细胞仅能存活3代。在KSFM中,培养细胞呈典型铺路石样外观,RT-PCR和Westernblotting结果显示细胞表达p63、角蛋白19以及involucrin。当培养基中加入胎牛血清后,细胞形态明显增大,呈扁平状;involucrin表达显著增强。结论：胎牛血清可抑制小鼠角膜上皮细胞增生、诱导其分化。     

Effect of hypoxia on the proliferation of murine cornea limbal epithelial progenitor cells in vitro

AIM: To investigate the effect of hypoxia on the proliferation of mouse corneal epithelial cells in vitro. METHODS:Mouse corneal epithelial cells(MCEs) were cultured in normoxia (210mL/L O2 and 50mL/L CO2) and hypoxia (20mL/L O2 and 50mL/L CO2), respectively. Colony forming efficiency (CFE) and cell proliferation were determined. The expression of corneal epithelial progenitor cell marker p63 and K19 was investigated by immunostaining. RESULTS: Normoxic colonies were smaller compared with colonies formed in hypoxia. CFE was (12.50±1.50)% in hypoxic cultures, which was similar compared with normoxia cultures [(11.13±1.86)%, P >0.05)]. Cell proliferation was enhanced in hypoxia. Progenitor markers p63 and K19 were expressed in most cells under both normoxic and hypoxic conditions. CONCLUSION: Murine limbal epithelial progenitor cells can be efficiently expanded in hypoxic conditions.     

兔角膜内皮、上皮及基质细胞体外培养扩增的研究

目的 建立角膜上皮、基质及内皮细胞体外培养扩增的简单稳定的方法，为组织工程化角膜的构建提供种子细胞。方法 内皮细胞与后弹力层在培养基中孵育后消化法获原代细胞，胰酶消化去除表层上皮后取角膜缘，组织块法培养角膜缘上皮细胞，基质细胞应用胶原酶消化法获原代培养，各细胞融合后胰酶消化依次传代培养。结果 原代内皮细胞4—5d融合成单层细胞，可连续传6—7代。上皮细胞1周左右生长融合，连续传3—4代后细胞形态改变。基质细胞接种6—7d后近融合，传代后增殖明显，可连续传10代。结论依据角膜组织特征选择合适的方法体外分离、培养角膜3种细胞成分，可获连续传代扩增的角膜细胞。     

Establishment of a corneal epithelial cell line spontaneously derived from human limbal cells

The objective of this study was to establish a spontaneously derived human corneal epithelial cell line from a normal human limbus that retains differentiation potential and proliferative properties under continuous cell culture. After 50 passages of epithelial cells obtained from human limbal tissue a cell line spontaneously emerged. The immortalized cells showed a cobblestone appearance and displayed dense microvilli on their apical cell surface membrane. Colony forming efficiency was 5-6% and population doubling time was 19.6 h. In the mRNA level, cytokeratin (CK) 3 and 12 were detected in this cell line. In the protein level, the cells expressed CK3, CK12, CK14, CK19, vimentin, and some other proteins such as F-actin and beta-tubulin and beta(1)-integrin. They lacked p63. The immortalized cells had a heteroploid karyotype, but did not exhibit tumorigenic features. When cultured on an air-liquid interface the cells could form stratified multilayer epithelia. In summary, all these results indicated that a new human corneal epithelial cell line was spontaneously established from normal limbal tissue through serial culture. This cell line would be useful for studies of corneal epithelial biology and reconstructive corneal tissue engineering.