以羊膜为载体体外培养大鼠角膜缘干细胞的实验研究

发布时间：2018-04-09 17:22

本文选题：角膜缘　切入点：干细胞　出处：《重庆医学》2016年23期

【摘要】：目的比较角膜缘干细胞(LSCs)在不同羊膜上的生长情况,寻求LSCs体外培养的最佳载体。方法取健康剖宫产孕妇胎盘的羊膜组织,分别制备保留上皮的新鲜羊膜、去除上皮的新鲜羊膜和冷冻羊膜。消化法分离大鼠LSCs后将其按相同浓度分别种植于3种事先准备好的羊膜载体和无载体孔中,分为4组。实验1组:保留上皮的新鲜羊膜组;实验2组:去除上皮的新鲜羊膜组;实验3组:去除上皮的冷冻羊膜组;对照组:无羊膜载体组。4组细胞分别作细胞凋亡检测,p63、PCNA免疫荧光检测,检测阳性细胞计数。结果培养第3天,3个实验组培养的细胞排列紧密,融合形成单层,其中可见大小不等的团块状集落,细胞大小基本一致,多呈圆球形或卵圆形。对照组培养的细胞排列相对稀疏,基本融合,形成单层,可见散在的、大小不等的桑葚样细胞集落,集落细胞的生长特征及形态特点与实验组相似。实验组的细胞凋亡率均低于对照组,p63阳性率和PCNA阳性率均高于对照组(P0.01),尤以实验1组变化最为明显(P0.01)。结论羊膜载体有利于体外培养大鼠LSCs的扩增,保留上皮的新鲜羊膜能更好地维持体外培养LSCs的特性,是体外角膜缘干细胞培养的理想载体。
[Abstract]:Objective to compare the growth of limbal stem cells (LSCs) on different amniotic membranes and to find the best carrier for LSCs culture in vitro.Methods the amniotic tissue of placenta of healthy pregnant women with cesarean section was taken, and the fresh amniotic membrane of epithelium was prepared, the fresh amniotic membrane and frozen amniotic membrane of epithelium were removed.After isolating rat LSCs by digestion method, it was seeded into three amniotic membrane carriers and non-carrier holes prepared in advance according to the same concentration, and were divided into 4 groups.Experimental group 1: fresh amniotic membrane with preserving epithelium, group 2: fresh amniotic membrane removing epithelium, group 3: frozen amniotic membrane group with epithelium removed;In the control group, the cells of group 4 without amniotic membrane carrier were detected for apoptosis and PCNA, and the number of positive cells was detected.Results on the third day of culture, the cells of the three experimental groups were arranged closely and fused into monolayers. Among them, there were clusters of different sizes, the size of the cells was basically the same, most of them were spherical or oval.The cells cultured in the control group were relatively sparse and fused to form a monolayer. The colony of mulberry like cells with different sizes was seen in the control group. The growth characteristics and morphological characteristics of the colony cells were similar to those of the experimental group.The apoptosis rate of the experimental group was lower than that of the control group. The positive rate of p63 and PCNA in the experimental group was higher than that in the control group (P 0.01), especially in the experimental group 1.Conclusion amniotic membrane carrier is beneficial to the amplification of rat LSCs in vitro and preserved epithelium fresh amniotic membrane can better maintain the characteristics of cultured LSCs in vitro. It is an ideal carrier for corneal limbal stem cell culture in vitro.
【作者单位】：贵州医科大学组织学与胚胎学教研室;
【分类号】：R329.2

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