不同鼠龄视网膜干细胞培养及生物学特性
发布时间：2018-12-17 05:10
【摘要】：目的：通过对SD不同鼠龄大鼠视网膜干细胞（RSCs，Retinal stem cells）培养及生物学特性鉴定，建立可靠的视网膜干细胞体外分离、培养和鉴定方法。 方法：（1）分别取健康SD大鼠鼠龄为出生后24小时、14天和21天的睫状体部（含色素层）于无血清、添加EGF、bFGF及B27的培养液中培养；同时用大鼠的视网膜部位的细胞培养作为阴性对照。 （2）采用机械酶消化法和组织块培养法分别对同一鼠龄的睫状体部位RSCs进行培养比较，采取每次随机选取20个不同的视野计数，比较两种方法培养的差别。采用SPSStatistics统计软件，进行统计分析。 （3）取不同鼠龄组的大鼠第1代RSCs，采取每次随机选取20个不同的视野计数，比较不同鼠龄组细胞培养的差别。采用SPSStatistics统计软件，进行统计分析。 （4）应用免疫荧光染色检测RSCs的神经干细胞特异性抗原神经巢蛋白(nestin)、GFAP表达。 （5）用胎牛血清对培养的第2代RSCs进行体外诱导分化，，并对分化后的细胞免疫荧光染色检测GFAP及MAP-2蛋白表达。 结果：（1）机械酶消化法能观察到RSCs细胞球数目和标准差为1.70±0.656，而组织块培养法RSCs细胞数目和标准差为0.30±0.470，采用SPSStatistics统计软件，两组比较P＜0.05(α=0.05)，差异有统计学意义，其中使用机械酶消化法培养的细胞数目多。 （2）从出生后24小时、14天和21天SD大鼠的睫状体部分离出来的RSCs经培养所得的细胞生长形态与神经干细胞形态相似，能形成悬浮生长的细胞团，且传代后重新形成细胞团；观测发现鼠龄为24小时、14天和21天SD大鼠RSCs细胞数目和标准差分别为1.7±0.657、1.25±0.550、1.2±0.523。培养RSCs的细胞数目24小时组与14天组和21天组比较P＜0.05(α=0.05)，差异有统计学意义，其中24小时鼠龄大鼠培养RSCs增殖的细胞数目多。 （3）RSCs免疫荧光鉴定，nestin表达呈阳性，GFAP表达阴性。而从视网膜部位提取的细胞不能形成悬浮生长的细胞团，免疫荧光鉴定，nestin反应呈阴性。 （4）RSCs分化后的细胞行免疫荧光鉴定既有MAP-2表达呈阳性细胞、也有GFAP表达阳性细胞。 4、结论：（1）大鼠睫状体边缘区（指含色素层）存在具有自我更新以及多方向分化潜能的RSCs，出生24h的大鼠睫状体边缘区中分离得来的RSCs比其余时期的较容易进行体外培养，说明鼠龄越小越容易培养视网膜干细胞。 （2）机械酶消化法比组织块法更容易培养视网膜干细胞。 （3）RSCs在胎牛血清的的诱导下可以分化为神经元样细胞和神经胶质样细胞。
[Abstract]:Aim: to establish a reliable method for the isolation, culture and identification of retinal stem cells (RSCs) isolated, cultured and identified in vitro by means of culture and identification of retinal stem cells (RSCs,Retinal stem cells) in different ages of SD rats. Methods: (1) the ciliary body (including pigmented layer) of healthy SD rats at 24 hours, 14 days and 21 days after birth were cultured in serum-free medium supplemented with EGF,bFGF and B27. At the same time, the cell culture of rat retina was used as negative control. (2) the RSCs of ciliary body of the same age was cultured and compared by mechanical enzyme digestion method and tissue mass culture method. 20 different visual fields were selected at random each time to compare the difference between the two methods. SPSStatistics statistical software was used for statistical analysis. (3) the first generation RSCs, of rats of different age groups were randomly selected 20 different visual field counts each time to compare the difference of cell culture in different age groups. SPSStatistics statistical software was used for statistical analysis. (4) Immunofluorescence staining was used to detect the expression of nestin (nestin), GFAP, a specific antigen of neural stem cells in RSCs. (5) fetal bovine serum was used to induce the differentiation of the second passage of RSCs in vitro, and the expression of GFAP and MAP-2 protein was detected by immunofluorescence staining. Results: (1) the number and standard deviation of RSCs cells were 1.70 卤0.656 by mechanical enzyme digestion and 0.30 卤0.470 by tissue mass culture. SPSStatistics software was used. There was significant difference between the two groups (P < 0.05 (伪 = 0.05), in which the number of cells cultured by mechanical enzyme digestion was more than that of the control group. (2) the RSCs isolated from the ciliary body of SD rats at 24 hours, 14 days and 21 days after birth had similar morphology to that of neural stem cells. It was found that the number and standard deviation of RSCs cells in SD rats were 1.7 卤0.657 卤1.25 卤0.550,1.2 卤0.523 at 24 hours, 14 days and 21 days, respectively. The number of RSCs cultured in 24 hours group was significantly higher than that in 14 day group and 21 day group (P < 0. 05). The number of RSCs proliferating cells in 24 hour old rats was more than that in 24 hour old rats. (3) the expression of nestin was positive and the expression of GFAP was negative in RSCs immunofluorescence assay. However, the cells extracted from the retina could not form the cell clusters of suspension growth, and the nestin reaction was negative. (4) the MAP-2 positive cells and GFAP positive cells were detected by immunofluorescence after RSCs differentiation. Conclusion: (1) there is RSCs, in the marginal ciliary area (including pigmented layer) with the potential of self-renewal and multi-directional differentiation. The RSCs isolated from the marginal ciliary area of 24 h old rats was easier to be cultured in vitro than that in the other period, which indicated that the younger the rat was, the easier it was to culture the retinal stem cells. (2) it is easier to culture retinal stem cells by mechanical enzyme digestion than tissue block method. (3) RSCs could differentiate into neuron-like cells and glial cells induced by fetal bovine serum.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2383700
[Abstract]:Aim: to establish a reliable method for the isolation, culture and identification of retinal stem cells (RSCs) isolated, cultured and identified in vitro by means of culture and identification of retinal stem cells (RSCs,Retinal stem cells) in different ages of SD rats. Methods: (1) the ciliary body (including pigmented layer) of healthy SD rats at 24 hours, 14 days and 21 days after birth were cultured in serum-free medium supplemented with EGF,bFGF and B27. At the same time, the cell culture of rat retina was used as negative control. (2) the RSCs of ciliary body of the same age was cultured and compared by mechanical enzyme digestion method and tissue mass culture method. 20 different visual fields were selected at random each time to compare the difference between the two methods. SPSStatistics statistical software was used for statistical analysis. (3) the first generation RSCs, of rats of different age groups were randomly selected 20 different visual field counts each time to compare the difference of cell culture in different age groups. SPSStatistics statistical software was used for statistical analysis. (4) Immunofluorescence staining was used to detect the expression of nestin (nestin), GFAP, a specific antigen of neural stem cells in RSCs. (5) fetal bovine serum was used to induce the differentiation of the second passage of RSCs in vitro, and the expression of GFAP and MAP-2 protein was detected by immunofluorescence staining. Results: (1) the number and standard deviation of RSCs cells were 1.70 卤0.656 by mechanical enzyme digestion and 0.30 卤0.470 by tissue mass culture. SPSStatistics software was used. There was significant difference between the two groups (P < 0.05 (伪 = 0.05), in which the number of cells cultured by mechanical enzyme digestion was more than that of the control group. (2) the RSCs isolated from the ciliary body of SD rats at 24 hours, 14 days and 21 days after birth had similar morphology to that of neural stem cells. It was found that the number and standard deviation of RSCs cells in SD rats were 1.7 卤0.657 卤1.25 卤0.550,1.2 卤0.523 at 24 hours, 14 days and 21 days, respectively. The number of RSCs cultured in 24 hours group was significantly higher than that in 14 day group and 21 day group (P < 0. 05). The number of RSCs proliferating cells in 24 hour old rats was more than that in 24 hour old rats. (3) the expression of nestin was positive and the expression of GFAP was negative in RSCs immunofluorescence assay. However, the cells extracted from the retina could not form the cell clusters of suspension growth, and the nestin reaction was negative. (4) the MAP-2 positive cells and GFAP positive cells were detected by immunofluorescence after RSCs differentiation. Conclusion: (1) there is RSCs, in the marginal ciliary area (including pigmented layer) with the potential of self-renewal and multi-directional differentiation. The RSCs isolated from the marginal ciliary area of 24 h old rats was easier to be cultured in vitro than that in the other period, which indicated that the younger the rat was, the easier it was to culture the retinal stem cells. (2) it is easier to culture retinal stem cells by mechanical enzyme digestion than tissue block method. (3) RSCs could differentiate into neuron-like cells and glial cells induced by fetal bovine serum.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
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相关期刊论文 前2条
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2 鄢文海,陈正跃,崔景彬,李开荣,王俊萍,王建枝,许予明,朱红灿;β淀粉样蛋白诱导胚胎大鼠神经干细胞凋亡[J];实用儿科临床杂志;2004年08期
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