大鼠角膜缘干细胞分离培养及其横向分化能力初探

Isolation and culture of rat limbal stem cells and research on trans differation capacity

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(P<0.05);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。

Objective To isolate and culture the rat limbal stem cells（ LSCs） in vitro,and investigate briefly their capacity for transdifferentiation into neural stem cells（ NSCs） with cytokine EGF,b FGF and RA. Methods LSCs derived from rats were cultured and identified by immunohistochemistry in vitro. LSCs were induced to differentiate into NSCs in the presence of EGF（ 20 ng / mL）,b FGF（ 10 ng / mL） and with or without of RA（ group 1 or group 2）（ 25 ng / mL） for seven days. Cultures without factors were used as control group. Then the neural marker Nestin of the coultured cells were measured by immunohistology staining. Furthermore,the positive cell rate was counted under microscope between the 2 groups and analyzed by statistical software. Results It showed that P63 was positive in LSCs. Nestin in both of the differentiation groups was positive at the rate of（ 77. 01 ± 6. 32） % and（ 84. 01 ± 5. 43） % respectively,of which the second group was higher than the first one（ P〈 0. 05）. However,it was negative in the control group. A band of Nestin protein from cells was detected by western blot assay. Conclusion LSCs are successfully isolated and cultured in vitro. LSCs could be induced to differentiate into NSCs in the presence of EGF and b FGF. Moreover,the differentiation capability is enhanced in the condition of RA.