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。     

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     

化学处理硅胶对角膜上皮细胞生长的影响

目前，人工角膜在眼科临床应用中存在着较多的并发症。Legeais和Renard认为人工角膜前表面如果能够上皮化将有利于植入物的长期稳定。硅胶是一种疏水的高分子材料，疏水性不利于细胞的黏附和生长。如果对其表面进行一定的处理，将有利于硅胶人工角膜与受体角膜的连接，从而减少人工角膜的术后并发症。改变材料表面特性的常用方法有低温等离子体法、表面修饰法、化学改性法和杂化改性法。我们选用酸溶液和过氧化氢溶液对硅胶进行一定的处理，体外观察细胞的生长情况，细胞质和细胞膜蛋白的表达，进而推测其对组织相容性的影响。     

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。     

不同培养方法对组织工程化小鼠角膜上皮构建影响的比较研究

目的:探讨滋养细胞在小鼠角膜上皮细胞复层化中的作用,并研究构建组织工程化角膜上皮的理想方法。方法:在Transwell气液界面培养系统中,分别采用接触滋养层培养法、分离滋养层培养法、复式滋养层培养法以及无滋养层培养法等4种方法进行组织工程化小鼠角膜上皮的重建。HE染色进行组织学观察,免疫荧光法检测p63、角蛋白19以及involucrin的表达。结果:接触滋养层培养法、分离滋养层培养法中,角膜上皮复层化为3-4层;复式滋养层培养法中,角膜上皮复层化达5-7层;而无滋养层培养法中,复层化仅为2-3层。复式滋养层培养法构建的小鼠角膜上皮,基底细胞层和基底细胞上层表达祖细胞标记p63和角蛋白19,角膜上皮全层均表达分化标记involucrin。结论:复式滋养层培养法为构建组织工程化小鼠角膜上皮的理想方法。     

角膜缘干细胞对外周血淋巴细胞活化和增殖作用的实验研究

目的 观察角膜缘干细胞对外周血淋巴细胞活化和增殖的抑制活性，并分析其机制。方法 通过植片技术分离兔角膜上皮细胞以及角膜缘干细胞。存两种细胞单层上，观察淋巴细胞埘丝裂原刀豆蛋白A(ConA)的增殖反应。另外，将两种细胞培养上清直接转移到由ConA刺激的淋巴细胞增殖系统以观察两种细胞释放。某些因子的抑制效应。结果 单层角膜缘干细胞对ConA刺激的外周血淋巴细胞增殖反应可以施加有效的抑制效应。角膜缘干细胞以及角膜上皮细胞的培养上清对ConA刺激的外周血淋巴细胞增殖系统，仅在72h分离上清显示有抑制效应。结论 角膜缘干细胞通过细胞交互作用和(或)释放某些抑制性因子对淋巴细胞的活化和增殖发挥潜在的抑制作用。     

羊膜对免角膜上皮细胞影响的实验研究

目的：观察羊膜对在其上皮面培养的免角膜上皮细胞增生的影响。方法：将免角膜上皮细胞原代培养后,接种于羊膜上皮面,并用ＭＴＴ自动比色法分别于接种后１、３、５、７天检测羊膜对免角膜上皮细胞增生的影响。结果：接种后１,３,５天,羊膜对免角以细胞有显示的促增生作用（Ｐ〈０．０５）。结论：羊膜有保进免角膜上皮细胞增生的作用,可用做角膜表结构重建的理想材料。     

羊膜对兔角膜上皮细胞影响的实验研究

目的 :观察羊膜对在其上皮面培养的兔角膜上皮细胞增生的影响。方法 :将兔角膜上皮细胞原代培养后 ,接种于羊膜上皮面 ,并用MTT自动比色法分别于接种后 1、3、5、7天检测羊膜对兔角膜上皮细胞增生的影响。结果 :接种后 1 ,3,5天 ,羊膜对兔角膜上皮细胞有明显的促增生作用 (P <0 0 5)。结论 :羊膜有促进兔角膜上皮细胞增生的作用 ,可用做角膜表面结构重建的理想材料。     

体外角膜上皮细胞凋亡的研究

为观察离体角膜上皮细胞经传代培养后细胞死亡的规律及确定其性质。本文应用流式细胞仪 ( FACStarplus)检测凋亡细胞的亚 2倍体 DNA以及细胞凋亡的形态学观察 ,对体外培养的每一代兔角膜缘上皮细胞的凋亡情况进行了观察及分析。结果显示 :角膜缘原代上皮细胞被检测出 7.0 0± 2 .2 3 %的凋亡细胞 ;从第 6代开始 ,角膜上皮细胞凋亡数目显著升高 ( P<0 .0 1) ;之后细胞凋亡数目急剧增加 ,第 8～ 9代后细胞出现集体“自杀”现象。透射电镜观察到这些细胞出现了核浓缩、胞膜发泡、凋亡小体等典型细胞凋亡的形态学改变。结论 :离体角膜缘上皮细胞因外部生长环境的改变 ,在培养到第 6代时出现了细胞凋亡现象。     

角膜上皮细胞的免疫调节网络

建立人角膜上皮细胞的原代和传代培养。在培养的上皮细胞单层上观察了上皮细胞对外周血淋巴细胞活性和功能的影响，并研究了上皮细胞与转化生长因子－β（ＴＧＦ－β）、白细胞介素－１（ＩＬ－１）和前列腺素Ｅ２（ＰＧＥ２）之间的相互作用。结果提示：在角膜上皮层以及周围组织存在着复杂且有多效性的细胞和细胞因子的免疫调节网络，这对于角膜的防御以及角膜移植片的存活具有一定的生物学意义。     

Effect of fetal bovine serum on the proliferation and differentiation of murine corneal epithelial cells in vitro

AIM: To investigate the effect of fetal bovine serum (FBS) on the proliferation and differentiation of murine corneal epithelial cells in vitro . METHODS: Mouse corneal epithelial cells (MCEs) were cultured in serum-free low-Ca²⁺ medium (KSFM) and KSFM supplemented with 100mL/L FBS, respectively. Population doublings (PDs) were determined. The expressions of corneal epithelial cell markers p63, keratin 19 (K19) and involucrin were investigated by RT-PCR and Western blotting analyses. RESULTS: Cells in KSFM were stably subcultured over 25 passages; however, none of the cell lines could pass P3 in KSFM with FBS. In KSFM, the cells showed typical cobblestone appearance and expressed p63, K19 and involucrin. After medium was supplemented with FBS, cells became homogeneous, large and squamous. Furthermore, both RT-PCR and Western blotting analyses showed that the expression of involucrin was increased significantly. CONCLUSION: FBS has effects of inhibiting proliferation and triggering differentiation of MCEs.     

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.     

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.     

Effect of human autologous serum and fetal bovine serum on human corneal epithelial cell viability, migration and proliferation in vitro

AIM: To analyze the concentration-dependent effects of autologous serum (AS) and fetal bovine serum (FBS) on human corneal epithelial cell (HCEC) viability, migration and proliferation. METHODS: AS was prepared from 13 patients with non-healing epithelial defects Dulbecco''s modified eagle medium/Ham’s F12 (DMEM/F12) with 5% FBS, 0.5% dimethyl sulphoxide (DMSO), 10 ng/mL human epidermal growth factor, 1% insulin-transferrin-selenium, then were incubated in serum media: DMEM/F12 supplemented by 5%, 10%, 15% or 30% AS or FBS. HCEC viability was analyzed using cell proliferation kit XTT, migration using a wound healing assay, proliferation by the cell proliferation enzyme-linked immunosorbent assay (ELISA) BrdU kit. Statistical analysis was performed using the generalized linear model, the values at 30% AS or 30% FBS were used as the baselines. RESULTS: HCEC viability was the highest at 30% AS or 15% FBS and the lowest at 10% AS or 30% FBS application. HCEC migration was the quickest through 30% AS or 30% FBS and the slowest through 5% AS or 5% FBS concentrations. Proliferation was the most increased through 15% AS or 5% FBS and the least increased through 30% AS or 30% FBS concentrations. HCEC viability at 10% and 15% AS was significantly worse (P=0.001, P=0.023) compared to baseline and significantly better at 15% FBS (P=0.003) concentrations. HCEC migration was significantly worse (P≤0.007) and HCEC proliferation significantly better (P<0.001) in all concentration groups compared to baseline. CONCLUSION: For the best viability of HCEC 30% AS or 15% FBS, for HCEC migration 30% AS or 30% FBS, for proliferation 15% AS or 5% FBS should be used. Therefore, we suggest the use of 30% AS in clinical practice.     

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.     

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。