【作者】 杨琨；

【导师】 董方田；

【作者基本信息】 北京协和医学院 ， 眼科学， 2011， 博士

【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(p<0.05)。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(p<0.05)。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。更多还原

【Abstract】 Objectives1. To simplify the procedure of primary cultivation and purification of retinal Muller cells in vitro.2. To study the mechanism and regulation underlying the activation of the latent neural stem cell properties of Muller cells.3. To study the roles of Wnt and Notch signaling in the activation of neural stem cell properties in Muller glia.Methods1. Eyeballs were enucleated from newborn rats and retinas were mechanically dissociated into small aggregates and cultured in DMEM/F12 (Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F12 Ham’s) medium containing 10%FBS (fetal bovine serum) for 8-10 days. After that, the floating reinal aggregates and debris were removed and the medium was changed every 2-3 days to get more putified cell population. The cells were passaged when they were confluent. Immunofluorescence assay was performed to identify the cultured Muller cells, and FACS(Fluorescence activated cell sorting) was carried out to determine the purity.2. Muller cells were dissociated using 0.25%Trypsin-0.05%EDTA and cultured in Serum Free Medium (DMEM/F12 containing lx N2 supplement,2x B27 supplement,2 mM L-glutamine,100 U/ml penicillin and 100μg/ml streptomycin) supplemented with 10 ng/ml bFGF(basic fibroblast growth factor) and 20 ng/ml EGF (epidermal growth factor) at a density of about 1 x 105 cells/cm2 for 3-5 days to generate neurospheres, refreshed the medium and grow factors every 2 days. Immunofluorescence assay, Real time RT-PCR and Western blotting were performed to test the expression of stem cell markers such as Nestin, Musashi-1 and Pax6 in the neurospheres, CCK-8 assay was to test the proliferation of the neurospheres. To examine the differentiation potential ability, neurospheres were shifted into the differentiation medium (DMEM/F12 containing 10%FBS, lx N2 supplement,2x B27 supplement,2mM L-glutamine,100 U/ml penicillin and 100μg/ml streptomycin) with the absence of EGF and bFGF, culture was continued for 5-7 days, immunofluorescence assay was to test the expression of GFAP in the redifferentiated cells.3. The mRNA levels of the components in Wnt and Notch pathway such as Fzdl, Fzd2, Lefl, Notch 1, Delta 1 and Hesl in the neurospheres were quantified by Real time RT-PCR analysis compared to which in the primary cultured Muller cells. During the dedifferentiation assay in Muller cells, Western blotting was performed to test the protein levels of Wnt2,β-catenin, Notch I, Pax6 and Nestin in d0, d1, d2, d3, d4 and d5.4. Pathway agonist (Wnt-3a, Notch1) and antagonist (Dkk-1, DAPT) were to be added to perturbed the Wnt and Notch signaling for studing their regulation effects in activating the neural stem cell properties in Muller glia. FACS was performed to test the Pax6 positive cells in the neurospheres. CCK-8 assay was to reveal the proliferating ability of the neurospheres by interfering the Wnt and Notch signaling.Results1. Cultured Muller cell had large cell bodies and relatively richer cytoplasma. About 97.2±1.43%of the cells were glutamine synthetase (GS) positive, and about 90.3±2.17%of the cells were Vimentin positive. FACS analysis showed that 99.7% of the cultured cells were GS positive after 3 passages.2. After 3-5 days cultured in the serum free medium, most of the Muller cells proliferated and formed neurospheres with an average size of 1OOμm in diameter. Immunofluorescence analysis show that, most of the neurospheres express the stem cell markers such as Nestin, Musashi-1 and Pax6. The rusults of Real time RT-PCR showed that, Pax6 and Nestin mRNA levels in neurospheres was about 5.57-and 3.98-fold compared to the Muller glia (p<0.05). Western blotting analysis showed that the protein levels of Pax6 and Nestin were also increased in the neurospheres. In the FACS assay, the positive cells of Pax6 and Nestin in the neurospheres were 80.2%and 60.4%versus 7.8%and 7.3%in the Muller glia. CCK-8 assay revealed that the neurospheres have self-renewing ability and can redifferentiate in vitro cultrue expressing GFAP.3. Real time RT-PCR analysis showed that, the mRNA levels of the components in the Wnt and Notch pathways were increased, Fzdl, Fzd2, Lefl, Notchl, Deltal and Hesl was 2.44-,2.82-,4.62-,3.24-,2.64-and 5.71-fold compared to the Muller glia (p<0.05). Western blotting analysis showed that, the protein levels of Wnt2, β-catenin, Notch 1, Pax6 and Nestin was increased accordingly from d0 to d5.4. In the Wnt-3a+Notch1 group, there were significant more and larger neurospheres, while there were less and smaller in the Dkk-1+DAPT group. FACS analysis showed that, the Pax6 positive cells in the Wnt-3a+Notch 1 group was 94.1%, the Wnt-3a group was 87.1%and the Notch1 group was 76.2%, which were higher than the conrol (63.7%), while the Dkk-1 group(38.4%) and the DAPT group (31.7%) were lower. CCK-8 assay showed that, the neurospheres proliferate more quickly in the Wnt-3a+Notch 1, besides, Wnt-3a and Notch 1 group were still both higher than the cells in control group, while in the Dkk-1 and the DAPT group, they proliferated more slowly compared to the control group.Conclusions1. The modified procedure intoduced in our experiment is a simple and practical method for culturing retinal Muller cells.2. Retinal Muller cells are potential stem cell source in the adlut retina. New born SD rats Muller cells can generate clonal stem cell neruospheres with the sitimulation of growth factors in vitro and display cardinal features of neural stem cells.3. The neurospheres derived from retinal Muller cells exhibite the neural stem cell characteristics and have the ability of self-renewing and redifferentiation.4. The Wnt signaling plays an important roles in the activation of the stem cell properties in Muller cells in concert with Notch.. Up regulation of both the signaling can facilitate the dedifferentiation process of dedifferentiation and also can promote the proliferation of the neurospheres derived from Muller glia.更多还原