不同鼠齡視網(wǎng)膜干細胞培養(yǎng)及生物學特性
發(fā)布時間：2018-12-17 05:10
【摘要】：目的：通過對SD不同鼠齡大鼠視網(wǎng)膜干細胞（RSCs，Retinal stem cells）培養(yǎng)及生物學特性鑒定，建立可靠的視網(wǎng)膜干細胞體外分離、培養(yǎng)和鑒定方法。 方法：（1）分別取健康SD大鼠鼠齡為出生后24小時、14天和21天的睫狀體部（含色素層）于無血清、添加EGF、bFGF及B27的培養(yǎng)液中培養(yǎng)；同時用大鼠的視網(wǎng)膜部位的細胞培養(yǎng)作為陰性對照。 （2）采用機械酶消化法和組織塊培養(yǎng)法分別對同一鼠齡的睫狀體部位RSCs進行培養(yǎng)比較，采取每次隨機選取20個不同的視野計數(shù)，比較兩種方法培養(yǎng)的差別。采用SPSStatistics統(tǒng)計軟件，進行統(tǒng)計分析。 （3）取不同鼠齡組的大鼠第1代RSCs，采取每次隨機選取20個不同的視野計數(shù)，比較不同鼠齡組細胞培養(yǎng)的差別。采用SPSStatistics統(tǒng)計軟件，進行統(tǒng)計分析。 （4）應用免疫熒光染色檢測RSCs的神經干細胞特異性抗原神經巢蛋白(nestin)、GFAP表達。 （5）用胎牛血清對培養(yǎng)的第2代RSCs進行體外誘導分化，，并對分化后的細胞免疫熒光染色檢測GFAP及MAP-2蛋白表達。 結果：（1）機械酶消化法能觀察到RSCs細胞球數(shù)目和標準差為1.70±0.656，而組織塊培養(yǎng)法RSCs細胞數(shù)目和標準差為0.30±0.470，采用SPSStatistics統(tǒng)計軟件，兩組比較P＜0.05(α=0.05)，差異有統(tǒng)計學意義，其中使用機械酶消化法培養(yǎng)的細胞數(shù)目多。 （2）從出生后24小時、14天和21天SD大鼠的睫狀體部分離出來的RSCs經培養(yǎng)所得的細胞生長形態(tài)與神經干細胞形態(tài)相似，能形成懸浮生長的細胞團，且傳代后重新形成細胞團；觀測發(fā)現(xiàn)鼠齡為24小時、14天和21天SD大鼠RSCs細胞數(shù)目和標準差分別為1.7±0.657、1.25±0.550、1.2±0.523。培養(yǎng)RSCs的細胞數(shù)目24小時組與14天組和21天組比較P＜0.05(α=0.05)，差異有統(tǒng)計學意義，其中24小時鼠齡大鼠培養(yǎng)RSCs增殖的細胞數(shù)目多。 （3）RSCs免疫熒光鑒定，nestin表達呈陽性，GFAP表達陰性。而從視網(wǎng)膜部位提取的細胞不能形成懸浮生長的細胞團，免疫熒光鑒定，nestin反應呈陰性。 （4）RSCs分化后的細胞行免疫熒光鑒定既有MAP-2表達呈陽性細胞、也有GFAP表達陽性細胞。 4、結論：（1）大鼠睫狀體邊緣區(qū)（指含色素層）存在具有自我更新以及多方向分化潛能的RSCs，出生24h的大鼠睫狀體邊緣區(qū)中分離得來的RSCs比其余時期的較容易進行體外培養(yǎng)，說明鼠齡越小越容易培養(yǎng)視網(wǎng)膜干細胞。 （2）機械酶消化法比組織塊法更容易培養(yǎng)視網(wǎng)膜干細胞。 （3）RSCs在胎牛血清的的誘導下可以分化為神經元樣細胞和神經膠質樣細胞。
[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 鹵0.656 by mechanical enzyme digestion and 0.30 鹵0.470 by tissue mass culture. SPSStatistics software was used. There was significant difference between the two groups (P < 0.05 (偽 = 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 鹵0.657 鹵1.25 鹵0.550,1.2 鹵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.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329
本文編號：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 鹵0.656 by mechanical enzyme digestion and 0.30 鹵0.470 by tissue mass culture. SPSStatistics software was used. There was significant difference between the two groups (P < 0.05 (偽 = 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 鹵0.657 鹵1.25 鹵0.550,1.2 鹵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.
【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329
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相關期刊論文 前2條
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2 鄢文海,陳正躍,崔景彬,李開榮,王俊萍,王建枝,許予明,朱紅燦;β淀粉樣蛋白誘導胚胎大鼠神經干細胞凋亡[J];實用兒科臨床雜志;2004年08期
本文編號：2383700
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