miR-184对神经干细胞增殖分化的影响及其分子机制
发布时间：2018-09-01 11:39
【摘要】：研究背景神经干细胞(Neural Stem Cells,NSCs)是一类具有增殖和多向分化能力的细胞。哺乳动物中,NSCs主要存在于侧脑室的脑室下区(SVZ)及海马的颗粒细胞下层(SGZ),研究表明NSCs具有再生及修复中枢神经系统损伤的功能,但目前仍不十分清楚NSCs增殖及定向分化机制,因此有必要进一步探究NSCs的增殖分化机制。MicroRNA(miRNA)是一类长约18～22 nt小单链RNA分子,通过miRNA 5'端与其靶分子mRNA 3'端的UTR序列特异互补结合后,对mRNA起负调控作用。成熟miR-184在细胞内的主要表达形式为单链miR-184-3P,目前对miR-184在神经组织细胞中的作用少有研究。本研究通过构建pHBLV-U6-miR-184/miR-184-shRNA-ZsGreen基因过表达及干扰慢病毒载体,并感染体外培养的NSCs,使miR-184在细胞内过表达或沉默,进而探究miR-184对神经干细胞增殖分化的影响以及miR-184与Notch信号通路相互作用的分子机制。研究目的1.构建miR-184基因过表达及干扰慢病毒载体;2.探究miR-184在NSCs中的目标靶基因;3.研究miR-184对NSCs增殖分化的影响及其分子机制;4.研究miR-184与Notch信号通路在NSCs增殖分化时的相互作用机制。研究方法1.通过RT-PCR技术扩增出miR-184基因和miR-184 shRNA基因,并分别克隆到pHBLV-U6-Scramble-ZsGreen质粒载体中,构建慢病毒表达载体。分离培养孕14天的胎鼠脑室下区(SVZ区)的神经干细胞并鉴定。2.实验细胞分为过表达对照组、miR-184过表达组、干扰对照组和miR-184干扰组,每组分别感染对应的慢病毒,并用终浓度为3 μg/mL的嘌呤霉素,进行抗性筛选,收集各组存活细胞继续培养至3代。通过RT-qPCR实验,进行miR-184过表达验证。3.采用Brdu细胞渗入实验,检测各组细胞增殖情况,记录各组Brdu+/DAPI+的比例。用神经干细胞分化培养液培养各组NSCs,细胞免疫荧光法检测NSCs分化为神经元细胞的情况,记录MAP2+/DAPI+比例。并检测NeuN蛋白和NeuNmRNA表达水平,以及Notch通路相关蛋白Hes 1、Hes5及其mRNA表达水平。4.通过 targetScan、iRTarBase、miRanda 等软件预测到 Numbl 作为 miR-184的潜在靶基因,经Western blotting法及RT-qPCR法进行NSCs胞内靶基因验证,并通过双荧光素酶报告基因系统进行验证。研究结果1.成功构建 pHBLV-U6-miR-184/miR-184-shRNA-ZsGreen 慢病毒表达载体。免疫荧光结果显示分离培养的细胞90%呈Nestin和Sox2双阳性。2.细胞成功感染慢病毒,抗性筛选培养后,细胞生长良好,RT-qPCR结果显示,miR-184过表达组的miR-184相对表达量是过表达对照组的67.63±7.53倍,差异具有统计学意义(P0.05)。3.Brdu细胞渗入实验等实验结果显示,与过表达对照组相比较,miR-184过表达组Brdu+/DAPI+比例是过表达对照组的1.47 ±0.05倍,MAP2+/DAPI+比例是过表达对照组的0.88±0.03倍,而与干扰对照组相比较,miR-184干扰组Brdu+/DAPI+比例是干扰对照组的0.84±0.03倍,MAP2+/DAPI+比例是干扰对照组的1.18±0.03倍。与过表达对照组比较,miR-184过表达组的NeuN蛋白及其mRNA相对表达量明显降低,分别是过表达对照组的0.76±0.02倍、0.75±0.07倍,而与干扰对照组相比较,miR-184干扰组则明显升高,分别是干扰对照组的1.22±0.01倍、1.54±0.05倍,以上差异均有统计学意义(P0.05)。与各自对照组比较,miR-184过表达组Hes1、Hes5蛋白及其mRNA相对表达量明显升高,miR-184干扰组则明显降低,差异有统计学意义(P0.05)。4.通过iRTarBase和miRanda等软件,预测到Numb1是miR-184潜在的靶基因。与过表达对照组相比较,miR-184过表达组的Numb1蛋白的相对表达量明显降低,是过表达对照组的0.73±0.07倍。与干扰对照组相比较,miR-184干扰组Numb1蛋白相对表达量则升高,是干扰对照组的1.30±0.05倍。差异均有统计学意义(P0.05)。此外,miR-184过表达组Numb1 mRNA的表达水平是过表达对照组的0.98±0.07倍,差异无统计学意义(t=0.593,P=0.613)。结果表明miR-184可抑制Numb1蛋白的翻译过程,而不改变Numb1 mRNA的表达水平。而且双荧光素酶实验结果进一步表明Numb1是miR-184的一个靶基因。
[Abstract]:BACKGROUND Neural Stem Cells (NSCs) are a class of cells with the ability of proliferation and multidifferentiation. In mammals, NSCs mainly exist in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus. Studies have shown that NSCs have the function of regeneration and repair of central nervous system injury, but it is still not very clear at present. It is necessary to explore the mechanism of proliferation and differentiation of Chu NSCs. MicroRNA (microRNA) is a small single-stranded RNA molecule with a length of about 18-22 nt, which plays a negative role in regulating the expression of mRNA through the specific complementary binding of the UTR sequence of the 5'end of microNA with the 3'end of its target molecule mRNA. In this study, we constructed pHBLV-U6-Mi-184/Mi-184-shRNA-ZsGreen gene overexpression and interfered with lentiviral vectors, and infected NSCs cultured in vitro to overexpress or silence microRNA-184 in cells, thus exploring the effects of microRNA-184 on neural stem cell proliferation. Objective 1. To construct an overexpression of microRNAs-184 and interfere with lentiviral vectors; 2. To explore the target genes of microRNAs-184 in NSCs; 3. To study the effects of microRNAs-184 on proliferation and differentiation of NSCs and its molecular mechanism; 4. To study the proliferation of microRNAs-184 and Notch signaling pathways in NSCs. Methods 1. Mi-184 gene and Mi-184 shRNA gene were amplified by RT-PCR and cloned into pHBLV-U6-Scramble-ZsGreen plasmid vector to construct Lentivirus Expression vector. Neural stem cells in the subventricular zone (SVZ) of fetal rats at 14 gestational days were isolated and cultured and identified as overexpressed. 2. Mi-184 overexpression group, interfering control group and interfering group were infected with lentiviruses respectively, and purinomycin with final concentration of 3 ug/mL was used for resistance screening. Survival cells of each group were collected and cultured for 3 generations. RT-qPCR was used to verify the overexpression of Mi-184. 3. Brdu cell infiltration test was used to detect each group. The ratio of Brdu+/DAPI+ was recorded. NSCs were cultured in the differentiation medium of neural stem cells. The differentiation of NSCs into neurons was detected by immunofluorescence. The ratio of MAP2+/DAPI+ was recorded. The expression levels of NeuN protein and NeuN mRNA, Notch pathway related proteins Hes 1, Hes 5 and their mRNA were detected. Numbl was predicted to be a potential target gene for microRNA184 by targetScan, iRTarBase, and microRNAanda. The intracellular target gene of NSCs was verified by Western blotting and RT-qPCR, and was verified by a double luciferase reporter gene system. Results 1. The expression of pHBLV-U6-microRNA184/microRNA184-shRNA-ZsGreen lentiviruses was successfully constructed. Immunofluorescence assay showed that 90% of the cultured cells were Nestin and Sox2 positive. 2. Lentiviruses were successfully infected and the cells grew well after resistance screening. RT-qPCR results showed that the relative expression of microRNA-184 in the overexpression group was 67.63 (+7.53) times higher than that in the overexpression control group, and the difference was statistically significant (P 0.05). 3. Brdu was fine. Cell penetration test and other experimental results showed that the Brdu+/DAPI+ ratio in the overexpression group was 1.47+0.05 times higher than that in the overexpression control group, and the MAP2+/DAPI+ ratio was 0.88+0.03 times higher than that in the overexpression control group. Compared with the interference control group, the Brdu+/DAPI+ ratio in the interference group was 0.84+0.03 times higher than that in the interference control group, and MA was 0.84+0.03 times higher than that in the overexpression control group. The ratio of P2+/DAPI+ was 1.18 +0.03 times higher than that of the interference control group. Compared with the over-expression control group, the relative expression of NeuN protein and its mRNA in the over-expression group decreased significantly, which were 0.76 +0.02 times and 0.75 +0.07 times higher than that of the over-expression control group, respectively. Compared with the interference control group, the interference group of microwave-184 increased significantly, which was 1.2 times higher than that of the interference control group. Compared with the control group, the relative expression of Hes1, Hes5 protein and mRNA in the overexpression group of microRNA184 increased significantly, while that in the interference group of microRNA184 decreased significantly (P 0.05). 4. Numb1 was predicted to be potential microRNA184 by iRTarBase and microRNAanda software. Compared with the overexpression control group, the relative expression of Numb1 protein in the overexpression group of Mi-184 was significantly lower than that in the overexpression control group, which was 0.73 (+ 0.07) times. Compared with the interference control group, the relative expression of Numb1 protein in the interference group of Mi-184 was increased, which was 1.30 (+ 0.05) times higher than that in the interference control group. The expression level of Numb1 mRNA in the over-expressed group was 0.98 [0.07] times higher than that in the over-expressed control group (t = 0.593, P = 0.613). The results showed that the translation process of Numb1 protein was inhibited by microRNA184, but the expression level of Numb1 mRNA was not changed. Furthermore, the results of double luciferase assay showed that Numb1 was a target gene of microRNA184.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2
本文编号：2217088
[Abstract]:BACKGROUND Neural Stem Cells (NSCs) are a class of cells with the ability of proliferation and multidifferentiation. In mammals, NSCs mainly exist in the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone (SGZ) of the hippocampus. Studies have shown that NSCs have the function of regeneration and repair of central nervous system injury, but it is still not very clear at present. It is necessary to explore the mechanism of proliferation and differentiation of Chu NSCs. MicroRNA (microRNA) is a small single-stranded RNA molecule with a length of about 18-22 nt, which plays a negative role in regulating the expression of mRNA through the specific complementary binding of the UTR sequence of the 5'end of microNA with the 3'end of its target molecule mRNA. In this study, we constructed pHBLV-U6-Mi-184/Mi-184-shRNA-ZsGreen gene overexpression and interfered with lentiviral vectors, and infected NSCs cultured in vitro to overexpress or silence microRNA-184 in cells, thus exploring the effects of microRNA-184 on neural stem cell proliferation. Objective 1. To construct an overexpression of microRNAs-184 and interfere with lentiviral vectors; 2. To explore the target genes of microRNAs-184 in NSCs; 3. To study the effects of microRNAs-184 on proliferation and differentiation of NSCs and its molecular mechanism; 4. To study the proliferation of microRNAs-184 and Notch signaling pathways in NSCs. Methods 1. Mi-184 gene and Mi-184 shRNA gene were amplified by RT-PCR and cloned into pHBLV-U6-Scramble-ZsGreen plasmid vector to construct Lentivirus Expression vector. Neural stem cells in the subventricular zone (SVZ) of fetal rats at 14 gestational days were isolated and cultured and identified as overexpressed. 2. Mi-184 overexpression group, interfering control group and interfering group were infected with lentiviruses respectively, and purinomycin with final concentration of 3 ug/mL was used for resistance screening. Survival cells of each group were collected and cultured for 3 generations. RT-qPCR was used to verify the overexpression of Mi-184. 3. Brdu cell infiltration test was used to detect each group. The ratio of Brdu+/DAPI+ was recorded. NSCs were cultured in the differentiation medium of neural stem cells. The differentiation of NSCs into neurons was detected by immunofluorescence. The ratio of MAP2+/DAPI+ was recorded. The expression levels of NeuN protein and NeuN mRNA, Notch pathway related proteins Hes 1, Hes 5 and their mRNA were detected. Numbl was predicted to be a potential target gene for microRNA184 by targetScan, iRTarBase, and microRNAanda. The intracellular target gene of NSCs was verified by Western blotting and RT-qPCR, and was verified by a double luciferase reporter gene system. Results 1. The expression of pHBLV-U6-microRNA184/microRNA184-shRNA-ZsGreen lentiviruses was successfully constructed. Immunofluorescence assay showed that 90% of the cultured cells were Nestin and Sox2 positive. 2. Lentiviruses were successfully infected and the cells grew well after resistance screening. RT-qPCR results showed that the relative expression of microRNA-184 in the overexpression group was 67.63 (+7.53) times higher than that in the overexpression control group, and the difference was statistically significant (P 0.05). 3. Brdu was fine. Cell penetration test and other experimental results showed that the Brdu+/DAPI+ ratio in the overexpression group was 1.47+0.05 times higher than that in the overexpression control group, and the MAP2+/DAPI+ ratio was 0.88+0.03 times higher than that in the overexpression control group. Compared with the interference control group, the Brdu+/DAPI+ ratio in the interference group was 0.84+0.03 times higher than that in the interference control group, and MA was 0.84+0.03 times higher than that in the overexpression control group. The ratio of P2+/DAPI+ was 1.18 +0.03 times higher than that of the interference control group. Compared with the over-expression control group, the relative expression of NeuN protein and its mRNA in the over-expression group decreased significantly, which were 0.76 +0.02 times and 0.75 +0.07 times higher than that of the over-expression control group, respectively. Compared with the interference control group, the interference group of microwave-184 increased significantly, which was 1.2 times higher than that of the interference control group. Compared with the control group, the relative expression of Hes1, Hes5 protein and mRNA in the overexpression group of microRNA184 increased significantly, while that in the interference group of microRNA184 decreased significantly (P 0.05). 4. Numb1 was predicted to be potential microRNA184 by iRTarBase and microRNAanda software. Compared with the overexpression control group, the relative expression of Numb1 protein in the overexpression group of Mi-184 was significantly lower than that in the overexpression control group, which was 0.73 (+ 0.07) times. Compared with the interference control group, the relative expression of Numb1 protein in the interference group of Mi-184 was increased, which was 1.30 (+ 0.05) times higher than that in the interference control group. The expression level of Numb1 mRNA in the over-expressed group was 0.98 [0.07] times higher than that in the over-expressed control group (t = 0.593, P = 0.613). The results showed that the translation process of Numb1 protein was inhibited by microRNA184, but the expression level of Numb1 mRNA was not changed. Furthermore, the results of double luciferase assay showed that Numb1 was a target gene of microRNA184.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2
【参考文献】
相关期刊论文 前3条
1 叶玉勤;贺晓生;;内源性神经干细胞参与神经再生的分子影像学研究进展[J];中华神经医学杂志;2014年06期
2 石海杉;吴文;徐建兰;;内源性神经干细胞的研究进展[J];实用医学杂志;2013年19期
3 郭永坤;张业森;刘春颖;戴宜武;黄瑾;姚慧;吴炳山;姚学勤;杨志军;徐如祥;;hTfR报告基因慢病毒载体构建及其在神经干细胞的表达研究[J];中华神经医学杂志;2013年05期
，本文编号：2217088
本文链接：https://www.wllwen.com/yixuelunwen/jichuyixue/2217088.html
