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?à1.50)% in hypoxic cultures,which was similar compared with normoxia cultures [(11.13?à1.86)%,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.·

Transplantation of human limbal cells cultivated on amniotic membrane for reconstruction of rat corneal epithelium after alkaline burn

Background The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model. Methods Human limbal cells were isolated and cultivated in vitro. Cytokertins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn. Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining.Results On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days’ labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no neovascularization. A majority of epithelial cells of the reconstructed cornea were positive to antihuman nuclear antibody and cells expressing K3 were found mainly in superfacial epithelium.Conclusions Limbal stem cells can be cultivated in vitro: the cells are characterized by high proliferation and slow cycling and identified as p63/K19 positive and K3/K12 negative. During culture, some stem cells can proliferate and differentiate into mature cornea epithelial cells. Amniotic membrane is a suitable carrier for limbal stem cells. Transplantation of human limbal stem cells cultivated on amniotic membrane can functionally reconstruct rat cornea with limbal stem cell deficiency.

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。

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(英文)

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。

ROCK抑制剂Y-27632在角膜保存时对角膜缘干细胞活性的促进作用

背景 角膜缘干细胞（LSCs）对维持角膜上皮的稳定和角膜组织透明性有重要作用.Rho关联卷曲螺旋蛋白激酶（ROCK）抑制剂Y-27632能够促进人胚胎干细胞、角质上皮细胞等的增生,减少细胞凋亡.目的 探讨Y-27632对离体兔角膜保存和体外LSCs扩增能力的影响. 方法 在细胞培养基MEM中加入质量分数12.5％硫酸软骨素、质量分数10.0％低分子右旋糖酐、20.0 mg/L地塞米松、100 mg/L妥布霉素注射液、9.5 g/L Hepes,使用时添加0.375 mg/L L-谷氨酰胺,制备成角膜活性保存液.将新西兰大白兔的角膜组织片分别置于含或不含Y-27632的角膜活性保存液中保存4、7、14d,采用质量分数0.25％锥虫蓝和质量分数0.2％茜素红染色法观察角膜内皮细胞密度及形态,采用Giemsa染色法并使用Image J图像分析软件计数克隆球数量和LSCs活性,计算LSCs存活率和克隆形成效率. 结果 角膜片保存4d时,Y-27632保存液组和单纯角膜中期保存液组角膜内皮细胞形态无明显差别,角膜片保存7d时,Y-27632保存液组角膜内皮细胞形态仍保持规则的六边形,而单纯角膜中期保存液组角膜内皮细胞的细胞膜轻微皱缩,少数细胞体积变大.角膜片保存14 d时单纯角膜中期保存液组角膜内皮细胞可见较多的茜素红斑.单纯角膜中期保存液组角膜内皮细胞计数为（2 262±75）/mm2,Y-27632保存液组角膜内皮细胞计数为（2 425 ±95）/mm2,差异有统计学意义（P＜0.001）.新鲜角膜片和角膜片保存4d时,Y-27632保存液组和单纯角膜中期保存液组LSCs的克隆球均较大,其内细胞数较多,而保存7d和14 d时,与Y-27632保存液组比较,单纯角膜中期保存液组LSCs克隆球直径明显缩小.新鲜分离的角膜片和角膜片保存4d时,Y-27632保存液组与单纯角膜中期保存液组LSCs的克隆形成率和角膜上皮细胞存活率的差异均无统计学意义（均P＞0.05）;角膜片保存7d和14d时,角膜缘上皮细胞的活性率分别为（73.00±2.12）％和（56.00±0.71）％,明显高于单纯角膜中期培养液组的（66.00±4.00）％和（49.00±0.71）％,差异均有统计学意义（t=3.098,P=0.018;t=9.798,P=0.000）;角膜片保存7d和14 d时,Y-27632保存液组与单纯角膜中期保存液组LSCs的克隆形成效率分别为（11.05±0.21）％和（3.10±1.97）％,明显高于单纯角膜中期保存液组中的（2.05±1.20）％和（0.40±0.14）％,差异均有统计学意义（t=18.107,P=0.000;t=3.184,P=0.017）.结论角膜保存液中添加Y-27632可明显提高离体角膜保存的效果,同时可维持LSCs的活性及克隆形成能力.Y-27632可作为角膜保存液的有效添加成分。

培养的角膜缘上皮细胞增殖能力的动态研究

目的 探讨组织块培养角膜缘上皮细胞在不同时间以及第 2、3代培养的角膜缘上皮细胞早期 (6～ 8d)的增殖能力。方法 应用组织块培养法培养角膜缘上皮细胞 ,在d8、12、16、2 0、2 4用流式细胞仪检测其细胞周期 ,同时对组织块培养 16d的角膜缘上皮细胞进行传代 ,用同样方法检测第 2、3代细胞出现汇合时 (6～ 8d)的细胞周期。结果 组织块培养角膜缘上皮细胞增殖指数 (ProliferativeIndex ,PI)在高水平上呈下降趋势 (分别为 31 2 %、2 0 1%、17 6 %、15 6 %、13 4 % ) ;2、3代培养细胞早期的增殖指数较高 (分别为 30 3%、2 6 % )。结论 组织块培养的角膜缘上皮细胞增殖能力在 8～ 2 4d内在高水平上呈下降趋势 ,第 2、3代培养的细胞在培养早期的增殖能力也较高 。

复式滋养层培养法构建组织工程化小鼠角膜上皮的实验研究

目的探讨构建组织工程化小鼠角膜上皮的理想方法。方法在Transwell气液界面培养系统中,利用复式滋养层培养法构建组织工程化小鼠角膜上皮,HE染色进行组织学观察,分别利用RT-PCR、Western blot以及免疫荧光法检测上皮祖细胞标记p63、角蛋白19(K19)以及分化标记被膜素(involucrin)的表达。结果复式滋养层培养法构建可成功小鼠角膜上皮,上皮层数达5～7层。与RT-PCR和Western blot结果一致,免疫荧光结果显示上皮基底细胞层和基底细胞上层表达p63和K19,角膜上皮全层均表达involucrin。结论复式滋养层培养法可保持培养细胞的干细胞性质,是构建组织工程化小鼠角膜上皮的理想方法。

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(英文)

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。

水牛输卵管上皮细胞无血清培养的研究

以广西沼泽型水牛(Bubalus bubalis)输卵管为材料,比较研究不同取材方法、有无血清及生长因子对水牛输卵管上皮细胞培养的影响。试验采用了三种不同的取材方法。原代用含血清的培养液来培养,传代用添加生长因子的无血清复杂培养液和不添加生长因子的无血清基础培养液结合梯度减量无血清培养法和直接无血清培养法来培养并进行观察。结果表明,水牛输卵管上皮细胞可以在添加生长因子的无血清复杂培养液中生长、并且最长可维持56 d优于基础无血清培养的15 d;刮取法获得的细胞活率为95%优于消化法的70%和剪碎法的83%;细胞冷冻复苏率为83.6%;第5代的染色体数目为48条,这说明细胞依然保持正常的生物学特性和二倍体核型。

以兔去上皮浅层角膜为载体体外培养角膜缘上皮细胞的实验研究

目的建立以兔去上皮浅层角膜为载体体外培养兔角膜缘上皮细胞的培养方法,观察细胞生物学特性。方法用含5%胎牛血清的DMEM/F12(1∶1)培养液对兔角膜缘上皮细胞进行体外组织块法原代培养,待细胞形成单层后,传代种植于去上皮浅层角膜载体上。每天用倒置显微镜观察培养细胞的形态及生长情况,电镜观察培育细胞的超微结构并采用HE染色和抗增殖细胞核抗原(anti-proliferating cell nuclear antigen,PCNA)单克隆抗体免疫细胞化学染色对生长于去上皮浅层角膜载体上的细胞进行检测。结果原代培养时,48小时后可见组织块边缘有细胞生长游出,10天左右,细胞汇合成单层。传代种植于载体上,细胞24小时贴壁伸展,第5天可见部分区域细胞逐渐融合成片,11天左右细胞形成良好单层。细胞呈卵圆形或多边形,类似角膜上皮细胞;电镜下,可见细胞表面有许多微绒毛,细胞间有桥粒连接;免疫细胞化学染色显示部分细胞PCNA单克隆抗体呈阳性反应。结论含5%胎牛血清的DMEM/F12培养液适合角膜缘干细胞的体外培养,并能成功地在去上皮角膜浅层载体上培养出类似生理状态下的角膜上皮细胞。

培养的角膜干细胞羊膜片移植治疗角膜烧伤的实验和临床研究(英文)

目的 探讨离体培养的角膜干细胞增殖分化特性 ,观察干细胞羊膜移植片治疗角膜缘功能障碍的效果。方法 用克隆形成率 (colony formingefficiency ,CFE)、免疫组织化学染色等方法观察培养角膜干细胞的增殖力和分化表达状况。角膜干细胞培养后接种生长于羊膜上进行干细胞羊膜片移植 ,观察角膜上皮、组织浸润和新生血管情况。结果 原代和传第 1代角膜干细胞具有高增殖力 ,部分表达角质蛋白K3,角膜干细胞在羊膜上能够继续增殖分化为多层细胞结构的角膜上皮细胞层 ;角膜干细胞移植术后可以在角膜比存活 ,角膜比移植片紧密贴附 ,角膜上皮完整 ,经度基质炎性浸润 ,浅层新生血管减少 ,深层新生血管充盈减轻 ,睑球粘连得到松解。结论 角膜缘干细胞羊膜片移植治疗角膜缘功能障碍能够使患者获得进一步进行角膜移植的条件。