【摘要】：目的：通過(guò)對(duì)SD不同鼠齡大鼠視網(wǎng)膜干細(xì)胞（RSCs，Retinal stem cells）培養(yǎng)及生物學(xué)特性鑒定，建立可靠的視網(wǎng)膜干細(xì)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(shí)、14天和21天的睫狀體部（含色素層）于無(wú)血清、添加EGF、bFGF及B27的培養(yǎng)液中培養(yǎng)；同時(shí)用大鼠的視網(wǎng)膜部位的細(xì)胞培養(yǎng)作為陰性對(duì)照。 （2）采用機(jī)械酶消化法和組織塊培養(yǎng)法分別對(duì)同一鼠齡的睫狀體部位RSCs進(jìn)行培養(yǎng)比較，采取每次隨機(jī)選取20個(gè)不同的視野計(jì)數(shù)，比較兩種方法培養(yǎng)的差別。采用SPSStatistics統(tǒng)計(jì)軟件，進(jìn)行統(tǒng)計(jì)分析。 （3）取不同鼠齡組的大鼠第1代RSCs，采取每次隨機(jī)選取20個(gè)不同的視野計(jì)數(shù)，比較不同鼠齡組細(xì)胞培養(yǎng)的差別。采用SPSStatistics統(tǒng)計(jì)軟件，進(jìn)行統(tǒng)計(jì)分析。 （4）應(yīng)用免疫熒光染色檢測(cè)RSCs的神經(jīng)干細(xì)胞特異性抗原神經(jīng)巢蛋白(nestin)、GFAP表達(dá)。 （5）用胎牛血清對(duì)培養(yǎng)的第2代RSCs進(jìn)行體外誘導(dǎo)分化，，并對(duì)分化后的細(xì)胞免疫熒光染色檢測(cè)GFAP及MAP-2蛋白表達(dá)。 結(jié)果：（1）機(jī)械酶消化法能觀察到RSCs細(xì)胞球數(shù)目和標(biāo)準(zhǔn)差為1.70±0.656，而組織塊培養(yǎng)法RSCs細(xì)胞數(shù)目和標(biāo)準(zhǔn)差為0.30±0.470，采用SPSStatistics統(tǒng)計(jì)軟件，兩組比較P＜0.05(α=0.05)，差異有統(tǒng)計(jì)學(xué)意義，其中使用機(jī)械酶消化法培養(yǎng)的細(xì)胞數(shù)目多。 （2）從出生后24小時(shí)、14天和21天SD大鼠的睫狀體部分離出來(lái)的RSCs經(jīng)培養(yǎng)所得的細(xì)胞生長(zhǎng)形態(tài)與神經(jīng)干細(xì)胞形態(tài)相似，能形成懸浮生長(zhǎng)的細(xì)胞團(tuán)，且傳代后重新形成細(xì)胞團(tuán)；觀測(cè)發(fā)現(xiàn)鼠齡為24小時(shí)、14天和21天SD大鼠RSCs細(xì)胞數(shù)目和標(biāo)準(zhǔn)差分別為1.7±0.657、1.25±0.550、1.2±0.523。培養(yǎng)RSCs的細(xì)胞數(shù)目24小時(shí)組與14天組和21天組比較P＜0.05(α=0.05)，差異有統(tǒng)計(jì)學(xué)意義，其中24小時(shí)鼠齡大鼠培養(yǎng)RSCs增殖的細(xì)胞數(shù)目多。 （3）RSCs免疫熒光鑒定，nestin表達(dá)呈陽(yáng)性，GFAP表達(dá)陰性。而從視網(wǎng)膜部位提取的細(xì)胞不能形成懸浮生長(zhǎng)的細(xì)胞團(tuán)，免疫熒光鑒定，nestin反應(yīng)呈陰性。 （4）RSCs分化后的細(xì)胞行免疫熒光鑒定既有MAP-2表達(dá)呈陽(yáng)性細(xì)胞、也有GFAP表達(dá)陽(yáng)性細(xì)胞。 4、結(jié)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(lái)的RSCs比其余時(shí)期的較容易進(jìn)行體外培養(yǎng)，說(shuō)明鼠齡越小越容易培養(yǎng)視網(wǎng)膜干細(xì)胞。 （2）機(jī)械酶消化法比組織塊法更容易培養(yǎng)視網(wǎng)膜干細(xì)胞。 （3）RSCs在胎牛血清的的誘導(dǎo)下可以分化為神經(jīng)元樣細(xì)胞和神經(jīng)膠質(zhì)樣細(xì)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(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 吳小影;張荻;劉雙珍;;大鼠視網(wǎng)膜神經(jīng)元的原代培養(yǎng)方法[J];國(guó)際眼科雜志;2008年01期

2 鄢文海,陳正躍,崔景彬,李開(kāi)榮,王俊萍,王建枝,許予明,朱紅燦;β淀粉樣蛋白誘導(dǎo)胚胎大鼠神經(jīng)干細(xì)胞凋亡[J];實(shí)用兒科臨床雜志;2004年08期

本文編號(hào)：2383700