本文關(guān)鍵詞：大鼠視網(wǎng)膜Muller細胞的神經(jīng)干細胞特性及其Wnt和Notch信號通路調(diào)控機制的研究　出處：《北京協(xié)和醫(yī)學(xué)院》2011年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 視網(wǎng)膜 Müuller細胞 干細胞 Wnt Notch

【摘要】：目的 1.對視網(wǎng)膜Muller細胞的原代培養(yǎng)方法加以改良,建立簡單快捷的分離培養(yǎng)Muller細胞的方法。 2.通過體外去分化誘導(dǎo),探討大鼠視網(wǎng)膜Muller細胞干細胞潛能的激活條件及調(diào)控機制。 3.研究Wnt和Notch信號通路在Muller細胞去分化的調(diào)控作用。 方法 1.取新生5-7天SD大鼠,顯微鏡下分離視網(wǎng)膜,直接吹打成微小組織懸液后置于含10%胎牛血清(fetal bovine serum, FBS)的DMEM/F12(1:1)培養(yǎng)基中培養(yǎng),8-10天后行第一次換液,之后2-3天換液一次至細胞完全融合后進行傳代。免疫熒光組織化學(xué)檢測Muller細胞標志物谷氨酰胺合成酶(glutamine synthetase,GS)和波形蛋白(Vimentin)的表達進行細胞鑒定,使用FACS對所得細胞進行純度檢測。 2.取第3代視網(wǎng)膜Muller細胞,更換含DMEM/F 12(1:1),1*N2 supplement, 2*B27 supplement,20 ng/ml表皮生長因子(epidermal growth factor, EGF),10 ng/ml堿性成纖維細胞生長因子(basic fibroblast growth factor, bFGF)的無血清去分化培養(yǎng)基培養(yǎng)3-5天。倒置相差顯微鏡下觀察去分化后細胞的形態(tài)特征,免疫熒光組織化學(xué),Real time RT-PCR以及Western blotting檢測神經(jīng)干細胞標志物Nestin, Musashi-1以及視網(wǎng)膜干細胞標志物Pax6的表達情況。CCK-8法檢測去分化后細胞的增殖能力。更換含1*N2 supplement,2*B27 supplement,10%FBS的DMEM/F 12培養(yǎng)基對去分化后的細胞進行再分化誘導(dǎo),免疫熒光組織化學(xué)檢測膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein, GFAP)的表達情況。 3. Real time RT-PCR檢測Wnt和Notch信號通路的相關(guān)分子Fzdl,Fzd2,Lefl, Notchl, Delta 1和Hesl在Muller細胞去分化培養(yǎng)前后的mRNA水平,并于去分化培養(yǎng)的第0,1,2,3,4,5天分別收集細胞行Western blotting檢測Wnt2,β-catenin, Notch 1, Pax6和Nestin的蛋白水平。 4.使用通路激動劑(Wnt-3a, Notch 1)和抑制劑(Dkk-1, DAPT)對Muller去分化培養(yǎng)過程中的Wnt和Notch信號通路進行干預(yù),觀察Muller細胞去分化的情況,FACS檢測各組去分化前后視網(wǎng)膜干細胞標志物Pax6陽性細胞的百分率。CCK-8檢測各組中,神經(jīng)球細胞的自我增殖能力。 結(jié)果 1.原代培養(yǎng)的Muller細胞胞體狹長,胞漿豐富,免疫熒光檢測結(jié)果顯示,97.2±1.43%的細胞GS染色陽性,90.3±2.17%的細胞Vimentin染色陽性。流式細胞檢測顯示傳3代后的細胞99.7% GS表達陽性。 2.去分化培養(yǎng)3-5天后,大部分Muller細胞克隆生長成神經(jīng)球狀,免疫熒光組織化學(xué)檢測顯示,細胞球Nestin, Musashi-1及Pax6染色陽性。Real time RT-PCR的結(jié)果顯示,細胞球中Pax6的mRNA水平較Muller細胞增加了-5.57倍,Nestin的mRNA水平增加了-3.98倍,差異有統(tǒng)計學(xué)意義(p0.05)。Western blotting結(jié)果顯示,神經(jīng)球細胞中的Pax6和Nestin的蛋白表達水平較Muller細胞有明顯升高。FACS結(jié)果顯示,去分化前的Muller細胞中Pax6和Nestin的陽性率分別為7.8%和7.3%,去分化后的細胞為80.2%和60.4%。CCK-8檢測顯示,神經(jīng)球細胞可以自我增殖,同時經(jīng)再分化誘導(dǎo)培養(yǎng)后表達膠質(zhì)細胞標志物GFAP。 3. Real time RT-PCR結(jié)果顯示,神經(jīng)球細胞中Wnt和Notch通路相關(guān)基因的mRNA水平較Muller細胞明顯升高,其中Fzd1為-2.44倍,Fzd2為-2.82倍,Lef1為-4.62倍,Notch 1為-3.24倍,Delta1為-2.64倍,Hes1為-5.71倍,差異具有統(tǒng)計學(xué)意義(p0.05)。Western blotting結(jié)果顯示,從d0到d5,Wnt2,β-catenin, Notch 1, Pax6和Nestin蛋白的表達逐漸增強,通路相關(guān)蛋白Wnt2,P-catenin及Notch 1與干細胞標志物Pax6及Nestin的表達存在時間的一致性。 4.在添加Wnt-3a和Notch 1組中,神經(jīng)球的數(shù)量明顯增加,體積增大,添加Dkk-1和DAPT組中,神經(jīng)球數(shù)量較少,細胞增殖緩慢。FACS結(jié)果顯示,在Wnt-3a+Notchl組中,Pax6陽性的細胞為94.1%,Wnt-3a組中為87.1%, Notchl組中為76.2%,均較對照組的63.7%有所升高,在Dkk-1組中Pax6陽性細胞為38.4%, DAPT組中為31.7%,均較對照組明顯下降。CCK-8檢測結(jié)果顯示,在Wnt-3a+Notchl組,Wnt-3a組和Notchl組中,細胞增殖速度均較對照組快,在Dkk-1組和DAPT組中,細胞增殖速度較對照組下降。 結(jié)論 1.改良后的培養(yǎng)方法可以簡單快捷的分離純化視網(wǎng)膜Muller細胞。 2.生長因子的刺激可以在體外激活視網(wǎng)膜Miiller細胞的干細胞特性,Muller細胞很可能成為視網(wǎng)膜干細胞的一種潛在來源。 3. Muller細胞去分化后形成神經(jīng)球樣結(jié)構(gòu),表達神經(jīng)干細胞的標志物,并具備自我增殖和再分化的能力。 4. Wnt和Notch言號的上調(diào)可以促進Muller細胞向神經(jīng)干細胞去分化,同時促進神經(jīng)干細胞球的自我增殖。Wnt和Notch信號通路在Muller細胞去分化的過程中存在協(xié)同調(diào)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 鹵 1.43 % of cells were stained positive and 90.3 鹵 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 .

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R774.1

【參考文獻】

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1 陳永東;許迅;顧青;;SD大鼠M，

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