OECs上清液诱导NSCs分化前后膜蛋白质复合物的差异表达研究

发布时间：2018-01-15 02:11

  本文关键词：OECs上清液诱导NSCs分化前后膜蛋白质复合物的差异表达研究　出处：《湖南师范大学》2012年硕士论文　论文类型：学位论文

  更多相关文章： 嗅鞘细胞 神经干细胞 分化 神经元 嗅鞘细胞 神经干细胞 细胞膜 BN-PAGE 蛋白质复合物

【摘要】：第一章OECs培养及上清液对NSCs的诱导分化研究 目的：探索小鼠嗅鞘细胞(olfactory ensheathing cells,OECs)对神经干细胞(neural stem cells,NSCs)分化的影响。 方法：健康新生昆明小鼠10只,取大脑皮层组织,用含2%B27、1%N2、20ng/ml碱性成纤维生长因子(basic fibroblast growth factor, bFGF)和20ng/ml表皮生长因子(epidermal growth factor, EGF)的培养液对NSCs进行悬浮培养,每5-7天传代一次,纯化培养2-3代备用,免疫细胞化学法对NSCs进行巢蛋白(Nestin)鉴定；取新生昆明小鼠嗅球,0.25%的胰蛋白酶37℃消化,改良Nash差速贴壁培养,加阿糖胞苷抑制杂细胞生长,当细胞数量达到大约80%时吸出上清,加入新鲜培养基(无血清DMEM),反复冲洗3遍,收集条件培养液,每隔3天半量换液。-20℃保存条件培养液,免疫细胞化学法对OECs进行神经生长因子受体p75(nerve growth factor receptor p75, NGFR p75)鉴定；实验组NSCs加OECs条件培养液及无血清DMEM/F12液培养,对照组NSCs只加无血清DMEM/F12培养液培养,每隔3天换液。观察细胞生长及分化情况,神经丝蛋白200(neurofilament200,NF200)鉴定神经元,胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)鉴定胶质细胞。 结果：新生昆明小鼠大脑皮层中分离培养出NSCs,这些细胞具有长期增殖能力,免疫细胞化学鉴定显示Nestin染色阳性；新生昆明小鼠嗅球培养出OECs,7天NGFR p75染色见大量阳性细胞；对照组NSCs克隆球少部分贴壁,未见增殖分化,细胞逐渐萎缩,12天时细胞死亡,实验组2天后NSCs克隆球开始贴壁分化,7天时分化较明显,10天时免疫细胞化学染色,NF200染色显示NSCs克隆球大部分分化为神经元,GFAP染色显示NSCs克隆球少部分分化为胶质细胞。 结论：OECs上清液能诱导NSCs向神经元方向分化。 目的：探索小鼠嗅鞘细胞(olfactory ensheathing cells, OECs)上清液诱导神经干细胞(neural stem cells, NSCs)向神经细胞分化的分子机制,筛选出诱导分化的关键蛋白,为临床药物靶向治疗神经损伤提供实验依据。 方法：取新生昆明小鼠嗅球,0.25%的胰蛋白酶37℃消化,改良Nash差速贴壁培养,加阿糖胞苷抑制杂细胞生长,当细胞数量达到大约80%时吸出上清,加入新鲜培养基(无血清DMEM),反复冲洗3遍,收集条件培养液,每隔3天半量换液,-20℃保存条件培养液。免疫细胞化学法对OECs进行神经生长因子受体p75(nerve growth factorreceptor p75, NGFR p75)鉴定。C172.2小鼠神经干细胞系复苏、培养、传代,37℃快速融化、离心、吹匀,高糖改良Eagle培养基H-DMEM (含15%胎牛血清)培养,4-5天传代一次,扩增至第三代(P3)备用。实验组NSCs加OECs条件培养液及无血清DMEM液培养诱导分化1/2、1、4小时,对照组NSCs只加无血清DMEM培养液培养。应用超速离心法提取细胞膜,蓝绿温和胶凝胶电泳(Blue Native-PAGE, BN-PAGE)分离膜蛋白质复合物,胶内酶切差异蛋白点,电喷雾四级杆时间飞行质谱(ESI-Q-TOF-MS)鉴定各时间点之间差异蛋白质复合物的表达。将仪器自动产生的数据文件用Mascot软件(www. matrixscience.com)中的串联质谱数据库搜寻(MS/Ms Ion Search)功能进行搜索。 结果：新生小鼠嗅球培养出OECs,7天NGFR p75染色见大量阳性细胞;C17.2NSCs复苏后1天内可见细胞贴壁生长,培养4-5天后细胞大量生长,进行传代,传代2天后可见大量细胞,形态呈梭形,折光性强；BN-PAGE显示出11个条带,每个时间点胶条酶切这11个条带。ESI-Q-TOF-MS鉴定到79个肽段相匹配的蛋白质,其中53个蛋白有显著差别的表达,另外26个蛋白有相同丰度的表达。我们共鉴定到5个蛋白质复合物的差异表达。 结论：1.0ECs上清液诱导NSCs分化前期的4个时间点间膜蛋白质复合物的表达有差异； 2.我们发现5个差异蛋白质复合物：mRNP granule complex、 ATP synthase complex、eukaryotic translation initiation factor3(eIF-3) complex、BBS/CCT complex及Hsp90complex,通过各自功能的搜库查询,其中Hsp90复合物可能在分化过程中起关键启动作用,为筛选NSCs分化过程中的关键蛋白提供数据。
[Abstract]:First chapter OECs culture and supernatant fluid induced differentiation of neural stem cells Objective : To explore the effect of olfactory ensheathing cells ( OECs ) on the differentiation of neural stem cells . Methods : Ten healthy new Kunming mice were cultured in vitro . The cultured cells were cultured in DMEM / F12 medium . The cells were cultured in DMEM / F12 medium . The cells were cultured in DMEM / F12 medium without serum . Results : The cells were isolated and cultured in the cerebral cortex of Kunming mice . The cells had long - term proliferative ability , and the cells showed Nestin staining positive . The cells of the control group had no proliferation and differentiation . The cells died after 2 days . After 2 days in the experimental group , the cells died . After 2 days in the experimental group , the cells were differentiated into neurons . The expression of NF200 showed that the majority of the cells differentiated into neurons . The GFAP staining showed that there was a small partial differentiation of the subclones into glial cells . Conclusion : The supernatant of OECs can induce the differentiation of neural stem cells into neurons . Objective : To explore the molecular mechanism of the differentiation of neural stem cells and neural stem cells from olfactory ensheathing cells ( OECs ) from mouse olfactory ensheathing cells ( OECs ) into neural cells , and to screen out the key proteins for inducing differentiation and provide experimental basis for the targeted treatment of nerve injury . Methods : The cells were cultured in DMEM culture medium containing 15 % fetal bovine serum at 37 鈩，

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