【作者】 高美丽；

【导师】 史伟云；

【作者基本信息】 青岛大学 ， 眼科学， 2009， 硕士

【摘要】 目的建立人胎儿角膜上皮细胞体外原代培养和传代培养方法,优化人胎儿角膜上皮细胞的培养体系,为构建组织工程角膜提供上皮种子细胞。方法一、人胎儿角膜上皮原代和传代培养1、原代培养严格无菌操作获取人胎儿角膜片,采用组织块贴壁法(角膜缘部、中央部)、酶消化法原代培养人胎儿角膜上皮细胞。2、传代培养角膜缘部的细胞生长融合达80%以上,不同稀释浓度的胰酶/EDTA消化液消化传代分别接种于空板、小鼠3T3成纤维细胞饲养层、胎儿角膜基质细胞饲养层及HTK饲养层,培养液分别采用D/F12、小鼠3T3成纤维细胞条件培养液、胎儿角膜基质细胞条件培养液、HTK条件培养液。二、人胎儿角膜上皮细胞培养体系的条件优化1、三种条件培养液蛋白含量测定与细胞增殖检测将收集的小鼠3T3成纤维细胞条件培养液、胎儿角膜基质细胞条件培养液、HTK条件培养液使用BCA蛋白定量试剂盒测定样品的蛋白含量。设D/F12为对照组,MTT法检测3T3条件培养液、胎儿角膜基质细胞条件培养液、HTK条件培养液对人角膜上皮细胞系的增殖能力。2、牛角膜细胞破碎液蛋白含量测定与细胞增殖检测将收集的牛角膜上皮细胞、基质细胞及内皮细胞三种细胞破碎液原液使用BCA蛋白定量试剂盒测定样品的蛋白含量。设D/F12为对照组,MTT法检测含5%、10%、20%不同浓度的三种牛角膜细胞破碎液对人角膜上皮细胞系的增殖能力。三、人胎儿角膜上皮细胞的性质鉴定将P3代人胎儿角膜上皮细胞用K3/12抗体进行荧光染色,观察传代细胞是否还保持有角膜上皮细胞的特性。收集P3代人胎儿角膜上皮细胞总RNA,用角膜基质细胞作为阴性对照,检测K12、Pax6、K15、ABCG2和P75在mRNA水平上的表达情况。结果一、人胎儿角膜上皮细胞的生长情况观察1、原代培养角膜缘部组织块周围迁出的细胞以圆形或卵圆形为主,排列紧密,生长状态良好;中央部的角膜上皮细胞体积大,以大而扁平的类表层上皮细胞为主,生长状态差;消化法培养可观察到上皮消化成单细胞悬液,但接种后无细胞贴壁。2、细胞传代观察不同浓度消化液消化传代的细胞发现,采用0.05%胰酶+0.02%EDTA消化可观察到消化后的细胞大部分成单细胞状态,传代培养后细胞贴壁及增殖良好。接于HTK饲养层和胎儿角膜基质细胞饲养层上的细胞不易贴壁且细胞不增殖,接于3T3饲养层上的细胞克隆生长,增殖能力强,但继续传代后细胞老化,不再增殖,容易有角膜基质细胞污染。采用D/F12、HTK条件培养液及胎儿角膜基质细胞条件培养液培养,细胞增殖能力弱,逐渐老化,不能继续传代;采用3T3条件培养液培养,培养3天后,可见形态规则的卵圆形小细胞集落分布于不规则的大细胞间,细胞增殖能力强。传至P4代胎儿角膜上皮细胞仍有较强增殖能力,但小细胞明显减少。二、人胎儿角膜上皮细胞培养体系的条件优化1、三种条件培养液对人角膜上皮细胞增殖的影响用BCA蛋白定量试剂盒对3T3条件培养液、胎儿角膜基质细胞条件培养液、HTK条件培养液进行蛋白含量测定,结果显示3T3条件培养液组蛋白含量最高。MTT法检测结果显示3T3条件培养液可明显促进HCEC的增殖,其与胎儿角膜基质细胞条件培养液、HTK条件培养液和D/F12三组差异均有显著的统计学意义。2、三种牛角膜细胞破碎液对人角膜上皮细胞增殖的影响用BCA蛋白定量试剂盒对牛角膜上皮、基质及内皮细胞破碎液液体进行蛋白含量测定,结果显示牛角膜上皮细胞破碎液为914μg/ml,牛角膜基质细胞破碎液为953μg/ml,牛内皮细胞破碎细胞液为1252μg/ml。MTT法检测结果显示5%的牛上皮细胞破碎液、5%、10%牛基质细胞破碎液、10%牛内皮细胞破碎液均可明显促进HCEC的增殖,四组与D/F12之间差异有显著的统计学意义,四组间差异无统计学意义。三、人胎儿角膜上皮细胞的性质鉴定1、免疫荧光染色可见P3代的人胎儿角膜上皮细胞K3/12染色阳性。2、RT-PCR检测显示,P3代人胎儿角膜上皮细胞在mRNA水平上有K12、K15、Pax6、P75和ABCG2的高表达,而角膜基质细胞表达较弱。结论一、组织块贴壁法原代培养人胎儿角膜上皮细胞以及小鼠3T3成纤维细胞条件培养液传代培养方法基本建立;二、人胎儿角膜缘部上皮细胞增殖能力优于中央部;三、3T3细胞条件培养液能促进角膜上皮细胞的增殖。更多还原

【Abstract】 PurposeTo optimize and explore culture system of human fetal corneal epithelial cells (hFCECs),including the methods of primary culture and passage.Methods一、Primary culture and passage of hFCECs1、primary cultureTo obtain the human fetal cornea with strict asepsis.The hFCECs were cultured by sticking tissues piece method that the cornea were divided into the limbus and central tissue piece and digestive method,respectively.For digestive method,the digestive juice of 5mg/ml DispaseⅡ+0.25%trypsin was chosed.D/F12 with 10%fetal bovine serum and 100 U/ml penicillin and streptomycin were used in this study,the culture condition was 37℃and 5%CO2,and culture medium changed every three days2、Passage of hFCECsAfter more than 80%confluenced,cells from corneal limbus hFCECs were digested for 5-15min with different concentrations of trypsin/EDTA at 37℃,and then passaged at a ratio of 1:2.The cells were subcultured on empty plates,mouse 3T3 fibroblast feeder layer,fetal corneal stromal cell feeder layer or HTK feeder layer respectively.And D/F12, mouse 3T3 fibroblast conditioned medium,fetal corneal stromal cells conditioned medium or HTK conditioned medium with 10%FBS and 100 U/ml penicillin and streptomycin were used respectively as the culture medium.And the culture medium were changed every three days as well.二、The optimization of the culture condition for hFCECs1、The protein determination of three kinds of conditioned medium and their effects on proliferation of corneal epithelial cellsThe protein concentrations of mouse 3T3 fibroblast conditioned medium,fetal corneal stromal cells conditioned medium and HTK conditioned medium were determined using the BCA Protein Assay Kit following the manufacture’s instruction.Human corneal epithelial cell line(HCEC) were passaged and inoculated in 96-well plate at 3×10~3cells/well with DMEM/F12 including 10%FBS.The original culture medium were removed and changed with 3T3 conditioned medium,fetal corneal stromal cells conditioned medium,HTK conditioned medium and D/F12 respectively at the next day. The proliferation ability were detected by the method of MTT.2、The protein determination of bovine corneal cell lysates and their effects on the proliferation of corneal epithelial cellsThe protein concentrations of the lysates solution of bovine corneal epithelial cells, stromal cells and endothelial cells were determined using the BCA Protein Assay Kit following the manufacture’s instruction.Next day,the original medium were totally changed with D/F12 medium adding three kind of bovine corneal cell lysates which density is 5%,10%and 20%respectively.The proliferation ability were detected by the method of MTT as well.三、Identification of hFCECsThe K3/12 immunostaining of passaged corneal epithelial cells were performed in the present work..And the expression of K3,K12,K15,Pax6 and ABCG2 of passaged corneal epithelial cells were determined using RT-PCR..Results一、Observation of hFCECs culture1、primary cultureThe corneal epithelial cells growed from corneal limbus tissue piece were mainly round or oval,with a close arrangement and growed in good condition.But corneal epithelial cells growed from central tissue piece,were mainly large size and fiat-based type,like epidermal epithelial cells and growed in poor condition.Epithelial cells could be digested single cells suspension,but wasn’t adherent to the culture plate with digestive method.2、PassageThe effects of different concentrations of trypsin/EDTA in hFCECs passage were tested in this study.When the concentration of trypsin was 0.25%or 0.125%,the epithelial cells were injured seriously and then led to a low rate of cell adhesion and proliferation.When the concentration of EDTA was 0.04%,the epithelial cells was difficult to be digested as single cell suspension,and the adhension and proliferation ability were poor as well.However,when digested using 0.05%trypsin and 0.02%EDTA for 5-10min at 37℃,the epithelial cells could be digested perfect single cell suspension; and then passaged at the ratio of 1:2,a high adherent rate and fine proliferation could be observed. When hFCECs were passaged on the feeder layer of corneal stromal cells,the cells were hard to adhere and proliferation.When hFCECs were passaged on the feeder layer of 3T3 feeder layer,the cells can grow with strong proliferation ability;but ther cells were aged and no more proliferation when further passage were performed.When cultured in D/F12 medium with HTK conditioned medium,corneal stromal cells conditioned medium or without any addition,passaged hFCECs was hard to proliferate and continue to passage.However when cultured with 3T3 conditioned medium,some clusters of proliferating small cells could observed gradually,,and the cells had a good proliferated ability and can be further passaged.When passsged at P4,small cells decreased significantly although the proliferation ability still remained.二、The conditions optimization of hCECs culture1、The effects of three kinds of conditioned medium on the proliferation of fetal corneal epithelial cellsThe protein concentration of D/F12 medium,3T3 conditioned medium,Fetal corneal stromal cells conditioned medium and HTK conditioned medium were 798,985,844and 833μg/ml respectively.The MTT results shown that 3T3 conditioned medium could markedly promote the proliferation of HCECs.And the results of analysis of interclass variance shown that the difference of 3T3 conditioned medium group with the other three groups have significant statistical significance.2、The effects of three broken bovine corneal cell fluid on the proliferation of fetal corneal epithelial cellsThe protein concentration of bovine corneal epithelial,stromal and endothelial cell lysates were 914,953 and 1252μg/ml respectively.The MTT results shown that compared with the control group,5%bovine corneal epithelial cell lysates,5%and 10%bovine corneal stromal cell lysates and 10%bovine corneal endothelial cell lysates could promote the proliferation of HCECs significantly. And the results of analysis of interclass variance shown that the difference of the three corneal cells lysates groups with the control groups have significant statistical significance,but there no significant difference between the four corneal cells groups.三、Identification of hFCECs1、The passage of fetal corneal epithelial cells were stained with K3/12 antibodies,and positive staining could be seen in cytoplasm. 2、RT-PCR detection showed that K3,K12,K15,Pax6 and ABCG2 were highly expressed in hFCECs,but no or more weaker expression in Fetal corneal stromal cells.Conclusions1.The methods of sticking tissues piece to culture primary hFCECs and mouse 3T3 fibroblast conditioned medium to culture passage hFCECs were established;2.The Proliferative capacity of corneal limbus hFCECs is better than central hFCECs.3.3T3 conditioned medium could markedly promote the proliferation of epithelial cells;更多还原