大鼠角膜缘干细胞分离培养及其横向分化能力初探

发布时间：2018-01-08 05:08

本文关键词：大鼠角膜缘干细胞分离培养及其横向分化能力初探　出处：《局解手术学杂志》2016年04期 　论文类型：期刊论文

【摘要】：目的体外分离培养大鼠角膜缘干细胞(LSCs),并探讨在细胞因子EGF、b FGF联合RA作用下,LSCs体外横向分化为神经干细胞样细胞的能力。方法体外分离培养LSCs并进行p63免疫组化鉴定。在原代培养的LSCs中,设立2个诱导组进行分化实验,诱导组1加入EGF(20 ng/mL)和b FGF(10 ng/mL),诱导组2加入EGF(20 ng/mL)、b FGF(10 ng/mL)和RA(25 ng/mL),同时设立空白对照组。培养7 d后,收集各组细胞作神经元标志物Nestin的免疫组化检测及阳性细胞计数,收集各组细胞蛋白做蛋白免疫印迹分析各组Nestin蛋白表达情况,相关数据作统计学分析。结果免疫组化结果显示,体外分离培养的LSCs p63呈阳性,诱导分化的2组细胞Nestin呈阳性表达,阳性率分别为(77.01±6.32)%和(84.01±5.43)%,诱导组2的诱导率高于诱导组1,差异具有统计学意义(P0.05);对照组细胞Nestin呈阴性表达。蛋白免疫印迹结果显示,2个诱导组在240 k Da处可见Nestin蛋白条带,诱导组2 Nestin蛋白表达略高于诱导组1,对照组未见Nestin蛋白条带。结论成功分离培养大鼠LSCs,在细胞因子EGF、b FGF作用下,LSCs具有横向分化为神经干细胞样细胞的能力,同时联合使用RA有促进LSCs向神经干细胞样细胞分化的作用。
[Abstract]:Objective to isolate and culture rat limbal stem cells (LSCS) in vitro, and to investigate the effect of EGFB FGF combined with RA. The ability of LSCs to differentiate into neural stem cell-like cells in vitro. Methods LSCs was isolated and cultured in vitro and p63 was identified by immunohistochemistry. Two induction groups were set up to conduct differentiation experiment. Induction group 1 added EGF(20 ng / mL) and b FGF(10 ngr mLL, induction group 2 added EGF(20 ngr mL). B FGF(10 ngr mLL) and RA(25 ngr mLL were set up at the same time blank control group was set up and cultured for 7 days. The expression of Nestin was detected by immunohistochemistry and the positive cells were counted by immunoblotting. The expression of Nestin protein in each group was analyzed by Western blotting. Results Immunohistochemical results showed that LSCs p63 was positive in vitro and Nestin was positive in differentiated cells. The positive rates were 77.01 卤6.32% and 84.01 卤5.43%, respectively. The induction rate in group 2 was higher than that in group 1 (P 0.05). The expression of Nestin in the control group was negative, and the Western blot showed that the Nestin protein bands were found in the two induction groups at 240kDa. The expression of 2 Nestin protein in the induced group was slightly higher than that in the induced group 1, and no band of Nestin protein was found in the control group. Conclusion the isolated and cultured rat LSCs were successfully isolated and cultured in the cytokine EGF. B FGF could differentiate into neural stem cell like cells in the transverse direction, and the combination of RA could promote the differentiation of LSCs into neural stem cell-like cells.
【作者单位】：贵州医科大学组织学与胚胎学教研室;
【基金】：贵州省优秀科技教育人才省长专项资金项目(黔省专合字【2012】41号) 贵州省科技厅、贵阳医学院联合基金(黔科合LG字【2012】026号)
【分类号】：R772.2
【正文快照】： 角膜缘干细胞(limbal stem cells,LSCs)位于角膜缘上皮基底层,是一种成体干细胞,具有不断增殖分化和向心性移动的能力,能够修复替代衰老死亡的角膜上皮细胞,是角膜上皮再生的源泉,在角膜上皮的更新和角膜疾病的治疗中起着不可替代的作用[1]。角膜缘干细胞和神经干细胞均来源于

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