【作者】 屈雷； 王馨； 杨学义； 窦忠英；

【Author】 QU Lei, WANG Xin, YANG Xue-yi, DOU Zhong-ying(Shaanxi branch of national Stem Cell Engineering Research Centre, Northewest Sci-Tech University of Agricultureand Forestry, shaanxi, Yangling 712100)

【机构】 西北农林科技大学国家干细胞工程技术研究中心陕西分中心；

【摘要】 目的体外分离培养人胎儿角膜上皮细胞、基质细胞和内皮细胞。方法无菌获取30倒流产胎儿,共计60个角膜(其中4～6月龄22例,7～9月龄8例),分别比较消化法、组织块法以及消化法结合组织块法在分离和培养人胎儿角膜上皮、内皮、基质细胞的差异。结果角膜缘上皮用0.25%胰蛋白酶+0.02%EDTA消化后,4～6月龄和7～9月龄胎儿角膜在总细胞数和活细胞数上差异显著。培养时加入角膜缘组织块可以缩短细胞形成单层的时间,增加传代后细胞的活力。用dispase和胰蛋白酶消化角膜上皮,对获取细胞总数和细胞活力无明显差异,但dispase消化后可获得纯化的上皮细胞,并可明显增加细胞传代次数,最高传至10代。组织块法培养时,角膜上皮面向上和向下对上皮细胞培养没有明显差异,但角膜切成组织块后不能获得角膜上皮细胞,仅在全角膜培养时,可得到角膜上皮细胞。消化法获取的7～9月龄胎儿角膜内皮细胞数量较少,原代培养时与角膜基质细胞混合在一起生长,传代不能得到角膜内皮细胞。消化法结合组织块法在大多数情况下组织块边缘长出的细胞为梭形的成纤维细胞,极个别出现内皮细胞与成纤维细胞混居,传代培养4代后,内皮细胞不再生长,仅留成纤维细胞生长。胎儿角膜基质细胞组织块培养时,7～9天开始有细胞长出,继续培养5～6天可形成密集单层。消化法培养时有时会出现与角膜上皮细胞和内皮细胞一起生长的现象,经4～5次传代后可获得纯化的角膜基质细胞。另外,在组织块法培养角膜上皮、内皮细胞时,接种后,上皮细胞和内皮细胞先于角膜基质细胞从组织块边缘长出。结论胎儿角膜上皮细胞采用dispase消化法和全角膜组织贴壁法可获得纯化的角膜上皮,角膜内皮细胞体外纯化培养较难,而胎儿角膜基质细胞无论采用消化法还是组织块法都可得到纯化培养的目的。更多还原

【Abstract】 Objective TO Isolate and culture epithelial cells, endothelial cells and stromal cells from human fetus corneas in vitro .Methods 30 human fetus (that is 60 corneas) used in this study were received in the asepsis condition, in which among them ,44 corneas were from 4 to 6-month-old fetus, the other 16 were from 7 to 9-month-old fetus. The methods of the enzyme digestion, the explants culture and the enzyme digestion associated with the explants culture were used to separate epithelial cells, endothelial cells and stromal cells, respectively, and then, compare them and find out different Results When the corneal limbal epithelium were digested by 0.25% trypsin and 0.02%EDTA, the more dispersed cells and living cells from 7 to 9-month-old embryonic corneas were obtained than those from 4 to 6-month old embryonic corneas (P<0.05). It could shorten the period of forming a monolayer cells and increase the cells proliferation ability after being passaged that grown cells in a culture plate together with a limbal explants. There are not significantly different hi the dispersed cells and living cells digested by Dispase and trypsin (P>0.05), but the cells dispersed by Dispase could proliferate to more than 10 generations. In the explants culture, there are no different between the corneal epithelium sides up and down, it could only successfully culture when the cornea was not separated into small pieces. A fewer corneal endothelial cells from 7 to 9-month-old corneas grown together with stromal cells in the primary cells culture could not be pure after subcultured in vitro. At the same time, the corneal endothelium explants cultures after digested by trypsin could not also obtain pure endothelial cells. When the corneal stromal explants were cultured, the stromal cells could outgrow and form a monolayer after culture 12-15days. Moreover, the corneal stromal cells digested by trypsin would grow together with epithelial cells or endothelial cells, but the pure uncontaminated stromal cells could obtain after 4-5 passages. In addition, in the corneal explants culture, the corneal epithelial cells or endothelial cells outgrow more early 5-6 days than stromal ceUs. Conclusion The pure human fetus corneal epithelial cells could successfully be culture by the mean of digesting to Dispase or of directly culturing a whole fetus cornea; it is difficult to obtain pure corneal endothelial cells in our culture system; the corneal stromal cells could be pure by culturing the dispersed cells or corneal stromal explants.更多还原