大鼠视网膜Muller细胞的神经干细胞特性及其Wnt和Notch信号通路调控机制的研究

发布时间：2018-01-18 01:10

本文关键词：大鼠视网膜Muller细胞的神经干细胞特性及其Wnt和Notch信号通路调控机制的研究　出处：《北京协和医学院》2011年博士论文　论文类型：学位论文

更多相关文章： 视网膜 Müuller细胞 干细胞 Wnt Notch

【摘要】：目的 1.对视网膜Muller细胞的原代培养方法加以改良,建立简单快捷的分离培养Muller细胞的方法。 2.通过体外去分化诱导,探讨大鼠视网膜Muller细胞干细胞潜能的激活条件及调控机制。 3.研究Wnt和Notch信号通路在Muller细胞去分化的调控作用。方法 1.取新生5-7天SD大鼠,显微镜下分离视网膜,直接吹打成微小组织悬液后置于含10%胎牛血清(fetal bovine serum, FBS)的DMEM/F12(1:1)培养基中培养,8-10天后行第一次换液,之后2-3天换液一次至细胞完全融合后进行传代。免疫荧光组织化学检测Muller细胞标志物谷氨酰胺合成酶(glutamine synthetase,GS)和波形蛋白(Vimentin)的表达进行细胞鉴定,使用FACS对所得细胞进行纯度检测。 2.取第3代视网膜Muller细胞,更换含DMEM/F 12(1:1),1*N2 supplement, 2*B27 supplement,20 ng/ml表皮生长因子(epidermal growth factor, EGF),10 ng/ml碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)的无血清去分化培养基培养3-5天。倒置相差显微镜下观察去分化后细胞的形态特征,免疫荧光组织化学,Real time RT-PCR以及Western blotting检测神经干细胞标志物Nestin, Musashi-1以及视网膜干细胞标志物Pax6的表达情况。CCK-8法检测去分化后细胞的增殖能力。更换含1*N2 supplement,2*B27 supplement,10%FBS的DMEM/F 12培养基对去分化后的细胞进行再分化诱导,免疫荧光组织化学检测胶质纤维酸性蛋白(glial fibrillary acidic protein, GFAP)的表达情况。 3. Real time RT-PCR检测Wnt和Notch信号通路的相关分子Fzdl,Fzd2,Lefl, Notchl, Delta 1和Hesl在Muller细胞去分化培养前后的mRNA水平,并于去分化培养的第0,1,2,3,4,5天分别收集细胞行Western blotting检测Wnt2,β-catenin, Notch 1, Pax6和Nestin的蛋白水平。 4.使用通路激动剂(Wnt-3a, Notch 1)和抑制剂(Dkk-1, DAPT)对Muller去分化培养过程中的Wnt和Notch信号通路进行干预,观察Muller细胞去分化的情况,FACS检测各组去分化前后视网膜干细胞标志物Pax6阳性细胞的百分率。CCK-8检测各组中,神经球细胞的自我增殖能力。结果 1.原代培养的Muller细胞胞体狭长,胞浆丰富,免疫荧光检测结果显示,97.2±1.43%的细胞GS染色阳性,90.3±2.17%的细胞Vimentin染色阳性。流式细胞检测显示传3代后的细胞99.7% GS表达阳性。 2.去分化培养3-5天后,大部分Muller细胞克隆生长成神经球状,免疫荧光组织化学检测显示,细胞球Nestin, Musashi-1及Pax6染色阳性。Real time RT-PCR的结果显示,细胞球中Pax6的mRNA水平较Muller细胞增加了-5.57倍,Nestin的mRNA水平增加了-3.98倍,差异有统计学意义(p0.05)。Western blotting结果显示,神经球细胞中的Pax6和Nestin的蛋白表达水平较Muller细胞有明显升高。FACS结果显示,去分化前的Muller细胞中Pax6和Nestin的阳性率分别为7.8%和7.3%,去分化后的细胞为80.2%和60.4%。CCK-8检测显示,神经球细胞可以自我增殖,同时经再分化诱导培养后表达胶质细胞标志物GFAP。 3. Real time RT-PCR结果显示,神经球细胞中Wnt和Notch通路相关基因的mRNA水平较Muller细胞明显升高,其中Fzd1为-2.44倍,Fzd2为-2.82倍,Lef1为-4.62倍,Notch 1为-3.24倍,Delta1为-2.64倍,Hes1为-5.71倍,差异具有统计学意义(p0.05)。Western blotting结果显示,从d0到d5,Wnt2,β-catenin, Notch 1, Pax6和Nestin蛋白的表达逐渐增强,通路相关蛋白Wnt2,P-catenin及Notch 1与干细胞标志物Pax6及Nestin的表达存在时间的一致性。 4.在添加Wnt-3a和Notch 1组中,神经球的数量明显增加,体积增大,添加Dkk-1和DAPT组中,神经球数量较少,细胞增殖缓慢。FACS结果显示,在Wnt-3a+Notchl组中,Pax6阳性的细胞为94.1%,Wnt-3a组中为87.1%, Notchl组中为76.2%,均较对照组的63.7%有所升高,在Dkk-1组中Pax6阳性细胞为38.4%, DAPT组中为31.7%,均较对照组明显下降。CCK-8检测结果显示,在Wnt-3a+Notchl组,Wnt-3a组和Notchl组中,细胞增殖速度均较对照组快,在Dkk-1组和DAPT组中,细胞增殖速度较对照组下降。结论 1.改良后的培养方法可以简单快捷的分离纯化视网膜Muller细胞。 2.生长因子的刺激可以在体外激活视网膜Miiller细胞的干细胞特性,Muller细胞很可能成为视网膜干细胞的一种潜在来源。 3. Muller细胞去分化后形成神经球样结构,表达神经干细胞的标志物,并具备自我增殖和再分化的能力。 4. Wnt和Notch言号的上调可以促进Muller细胞向神经干细胞去分化,同时促进神经干细胞球的自我增殖。Wnt和Notch信号通路在Muller细胞去分化的过程中存在协同调控作用。
[Abstract]:Purpose 1 . The primary culture method of retinal M眉ller cells was modified to establish a simple and rapid method for the isolation and culture of muller cells . 2 . To explore the activation condition and mechanism of rat retinal M眉ller cell ' s potential by dedifferentiation induction in vitro . 3 . The role of Wnt and Notch signaling pathway in the differentiation of Muller cells was investigated . method 1 . After 5 - 7 days old SD rats were cultured in DMEM / F12 ( 1 : 1 ) medium containing 10 % fetal bovine serum ( FBS ) and cultured in DMEM / F12 ( 1 : 1 ) medium containing 10 % fetal bovine serum ( FBS ) . 2 . The expression of neural stem cell markers Nestin , Musashi - 1 , and retinal stem cells , Nestin , Musashi - 1 , and retinal stem cell markers were observed under the reversed phase contrast microscope . The expression of Nestin , Musashi - 1 and the marker of retinal stem cells were observed under the reversed phase contrast microscope . The expression of glial fiber acidic protein ( GFAP ) was detected by immunofluorescence histochemistry . 3 . Real time RT - PCR was used to detect the mRNA levels of Wnt and Notch signaling pathway , Fzd2 , Lefl , Notchl , Delta 1 and Hesl mRNA levels before and after the differentiation and culture of Muller cells . Western blotting was used to detect the protein levels of Wnt2 , 尾 - catenin , Notch 1 , 6 and Nestin on the 0 , 1 , 2 , 3 , 4 and 5 days of dedifferentiation culture . 4 . Using Wnt - 3a ( Notch 1 ) and inhibitor ( DLIF - 1 , DAPT ) to intervene the Wnt and Notch signaling pathway in the culture of M眉ller ' s dedifferentiation and observe the dedifferentiation of Muller cells , FACS was used to detect the percentage of the retinal stem cells and the percentage of the positive cells of the retinal stem cells before and after dedifferentiation . CCK - 8 detected the self - proliferative ability of the cells in each group . Results 1 . The cells of muller cells cultured in primary culture were elongated , the cytoplasm was abundant , and the results of immunofluorescence test showed that 93.2 卤 1.43 % of cells were stained positive and 90.3 卤 2 . 17 % were positive for vimentin stain . Flow cytometry showed that 99.7 % GS expression was positive after 3 generations . 2 . After 3 - 5 days of dedifferentiation culture , most of the muller cells were cloned into neurospheres . The results showed that the expression levels of the mRNA in the cells were increased by - 5.57 - fold and - 3.98 - fold higher than that of the muller cells . 3 . Real time RT - PCR showed that the mRNA levels of Wnt and Notch pathway related genes in neurosphere cells were significantly higher than those of Muller cells , with Fzd1 - 2.44 - fold , Fzd2 - 2.82 - fold , Lef1 - 4.62 - fold , Notch - 1 - 3.24 - fold , Delta1 - 2.64 - fold and Hes1 - 5.71 - fold , and the difference was - 2.64 - fold , Hes1 - 5.71 - fold . 4 . In the group of Wnt - 3a + Notchl group , the number of neurospheres increased significantly , the volume was increased , and the number of neurospheres increased . In the group of Wnt - 3a + Notchl , 76.1 % in Wnt - 3a + Notchl group , 76.2 % in the Notchl group , and 76.2 % in the Notchl group were significantly lower than those in the control group . Conclusion 1 . The modified culture method can separate and purify the retinal M眉ller cells simply and quickly . 2 . The stimulation of growth factors can activate the stem cell characteristics of the retinal Miiller cells in vitro , and the Muller cells are likely to be a potential source of retinal stem cells . 3 . The neural stem - like structure is formed after the M眉ller cells are dedifferentiated , which expresses the marker of neural stem cells and has the capability of self - proliferation and redifferentiation . 4 . The up - regulation of Wnt and Notch can promote the differentiation of neural stem cells and promote the self - proliferation of neural stem cells . Wnt and Notch signaling pathways have a synergistic effect in the process of dedifferentiation of muller cells .

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1

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