同型半胱氨酸对神经干细胞增殖的蛋白组学研究
发布时间：2019-02-16 09:51
【摘要】：目的 通过体外培养胚胎期SD大鼠神经干细胞(neural stem cells, NSCs),添加同型半胱氨酸(homocysteine, Hcy)进行干预,观察Hcy对神经干细胞增殖能力的影响,并进一步观察其增殖期蛋白表达谱的变化,探讨Hcy对神经干细胞增殖影响的机制；建立HuSH shRNA Plasmid介导的RNA干扰技术体系。 方法 采用无血清体外细胞培养方法,培养胚胎期大鼠NSCs,将NSCs分为四组：①正常对照组(normal control group)培养基中叶酸含量为4μg/mL,同型半胱氨酸含量为0μg/mL;②叶酸缺乏组(Folic acid-D group)培养基中叶酸含量为0.65μg/mL,同型半胱氨酸含量为0μg/mL;③同型半胱氨酸中度增高组(Hey-medium group)培养基中叶酸含量为4μg/mL,同型半胱氨酸含量为100μg/mL;④同型半胱氨酸高度增高组(Hcy-high group)培养基中叶酸含量为4μg/mL,同型半胱氨酸含量为300μg/mLc采用MTT法检测神经干细胞增殖能力,绘制生长曲线；计数神经球的数量及直径以检测神经球的结球能力；采用台盼蓝染色法检测神经干细胞活力；培养6天后收集增殖期细胞,提取细胞蛋白,进行双向电泳,经考马斯亮蓝染色后使用PD-Quest软件比对蛋白差异点,将筛选出的蛋白点进行质谱鉴定。采用Real-time PCR法检测经质谱鉴定蛋白mRNA表达水平。采用试剂盒检测神经干细胞内ATP含量、SOD活力、MDA水平、ROS含量；测定神经干细胞线粒体呼吸链酶复合物NADH-Q还原酶(CⅠ)、琥珀酸-Q还原酶(CⅡ)、细胞色素还原酶(CⅢ)及细胞色素氧化酶(CⅣ)的活性。采用HuSH shRNA Plasmid为载体建立RNA干扰技术体系。 结果 添加Hcy后,NSCs增殖水平受到抑制。MTT结果显示,添加Hcy后各时间点细胞活力较正常对照组有所降低,差异具有统计学意义(P0.05)。在Hcy添加组,神经球直径明显减少,与对照组比较差异具有统计学意义(P0.05)；但神经球数目各组间差异不明显(P0.05)。将神经球裂解后,计数神经干细胞数目发现,在两组Hcy添加组,细胞数目较对照组有所减少,且呈浓度依赖性趋势(P0.05)。经台盼蓝染色,发现各组间神经干细胞活力差异不明显(P0.05)。蛋白质组学结果显示,添加Hcy后神经干细胞增殖期蛋白表达谱发生了明显变化；我们从差异蛋白中挑选出7种蛋白进行质谱鉴定,成功鉴定6种蛋白；其中细胞色素b-c1复合体-亚基2和乌头酸水合酶为线粒体呼吸链和三羧酸循环涉及蛋白,提示Hcy可能是通过影响线粒体的功能而降低神经干细胞增殖的。Real-time PCR结果显示,细胞色素b-c1复合体-亚基2和乌头酸水合酶mRNA水平同样被Hcy抑制。采用试剂盒检测ATP含量发现Hcy降低了神经干细胞的能量合成(P0.05)；降低了SOD活性(P0.05),升高了MDA和ROS水平(P0.05)；且线粒体呼吸链复合酶Ⅰ～Ⅳ的活性均受到不同程度的抑制(P0.05)。成功建立经HuSH shRNA Plasmid介导的RNA干扰技术体系。 结论 本实验成功分离、培养了体外胚胎期SD大鼠NSCs,研究结果显示同型半胱氨酸可以抑制NSCs的增殖,降低神经球的结球能力,影响NSCs增殖期蛋白表达谱的水平；在mRNA和蛋白水平均降低细胞色素b-c1复合体-亚基2和乌头酸水合酶的表达；减少细胞内ATP的含量,增加活性氧自由基生成,降低其抗氧化能力,降低线粒体呼吸链复合酶Ⅰ～Ⅳ的活性。建立经HuSH shRNA Plasmid介导的RNA干扰技术体系。
[Abstract]:Purpose The effects of Hcy on the proliferation ability of neural stem cells were observed by the culture of neural stem cells (NSCs) in the embryonic stage SD rats in vitro, and the change of the expression profile of the protein in the proliferative phase was further observed. The mechanism of the effect of Hcy on the proliferation of neural stem cells was discussed, and the RNA interference technique mediated by HuSH shRNA Plasmaid was established. Department. Methods The non-serum in-vitro cell culture method was used to culture the NSCs in the embryonic stage, and the NSCs were divided into four groups: the content of the folic acid in the normal control group was 4.mu. g/ mL and the content of the same cysteine was 0. The folic acid content in the Folic acid-D group culture medium was 0.65 & mu; g/ mL, the content of the same cysteine was 0. mu. g/ mL, and the folic acid content in the medium-high group (Hy-medium group) medium of the same type of cysteine was 4. m u.g/ mL and the same cysteine content was 100. The content of folic acid in Hcy-high group (Hcy-high group) medium was 4.mu. g/ mL, the content of the same cysteine was 300. m u.g/ mLc, the proliferation ability of neural stem cells was detected by MTT method, the growth curve was drawn, and the number and diameter of the nerve ball were counted to detect the nerve ball. the activity of the neural stem cells is detected by a trypan blue staining method, the proliferation period cells are collected after 6 days of culture, the cell protein is extracted, two-way electrophoresis is carried out, the protein difference points are compared with the protein difference points by using the PD-Quest software ratio after the Coomassius brilliant blue staining, and the screened protein points are The identification of mRN by mass spectrometry was detected by real-time PCR. A. The level of ATP in the neural stem cells, the activity of SOD, the level of MDA and the content of ROS were detected by using the kit. The mitochondrial respiratory chain of the neural stem cells, NADH-Q reductase (C I), and the succinic acid-Q reduction, were determined. Enzyme (C II), Cytochrome Reductase (C 鈪，
本文编号：2424318
[Abstract]:Purpose The effects of Hcy on the proliferation ability of neural stem cells were observed by the culture of neural stem cells (NSCs) in the embryonic stage SD rats in vitro, and the change of the expression profile of the protein in the proliferative phase was further observed. The mechanism of the effect of Hcy on the proliferation of neural stem cells was discussed, and the RNA interference technique mediated by HuSH shRNA Plasmaid was established. Department. Methods The non-serum in-vitro cell culture method was used to culture the NSCs in the embryonic stage, and the NSCs were divided into four groups: the content of the folic acid in the normal control group was 4.mu. g/ mL and the content of the same cysteine was 0. The folic acid content in the Folic acid-D group culture medium was 0.65 & mu; g/ mL, the content of the same cysteine was 0. mu. g/ mL, and the folic acid content in the medium-high group (Hy-medium group) medium of the same type of cysteine was 4. m u.g/ mL and the same cysteine content was 100. The content of folic acid in Hcy-high group (Hcy-high group) medium was 4.mu. g/ mL, the content of the same cysteine was 300. m u.g/ mLc, the proliferation ability of neural stem cells was detected by MTT method, the growth curve was drawn, and the number and diameter of the nerve ball were counted to detect the nerve ball. the activity of the neural stem cells is detected by a trypan blue staining method, the proliferation period cells are collected after 6 days of culture, the cell protein is extracted, two-way electrophoresis is carried out, the protein difference points are compared with the protein difference points by using the PD-Quest software ratio after the Coomassius brilliant blue staining, and the screened protein points are The identification of mRN by mass spectrometry was detected by real-time PCR. A. The level of ATP in the neural stem cells, the activity of SOD, the level of MDA and the content of ROS were detected by using the kit. The mitochondrial respiratory chain of the neural stem cells, NADH-Q reductase (C I), and the succinic acid-Q reduction, were determined. Enzyme (C II), Cytochrome Reductase (C 鈪，
本文编号：2424318
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