miR-184對神經(jīng)干細(xì)胞增殖分化的影響及其分子機(jī)制
發(fā)布時間：2018-09-01 11:39
【摘要】：研究背景神經(jīng)干細(xì)胞(Neural Stem Cells,NSCs)是一類具有增殖和多向分化能力的細(xì)胞。哺乳動物中,NSCs主要存在于側(cè)腦室的腦室下區(qū)(SVZ)及海馬的顆粒細(xì)胞下層(SGZ),研究表明NSCs具有再生及修復(fù)中樞神經(jīng)系統(tǒng)損傷的功能,但目前仍不十分清楚NSCs增殖及定向分化機(jī)制,因此有必要進(jìn)一步探究NSCs的增殖分化機(jī)制。MicroRNA(miRNA)是一類長約18～22 nt小單鏈RNA分子,通過miRNA 5'端與其靶分子mRNA 3'端的UTR序列特異互補(bǔ)結(jié)合后,對mRNA起負(fù)調(diào)控作用。成熟miR-184在細(xì)胞內(nèi)的主要表達(dá)形式為單鏈miR-184-3P,目前對miR-184在神經(jīng)組織細(xì)胞中的作用少有研究。本研究通過構(gòu)建pHBLV-U6-miR-184/miR-184-shRNA-ZsGreen基因過表達(dá)及干擾慢病毒載體,并感染體外培養(yǎng)的NSCs,使miR-184在細(xì)胞內(nèi)過表達(dá)或沉默,進(jìn)而探究miR-184對神經(jīng)干細(xì)胞增殖分化的影響以及miR-184與Notch信號通路相互作用的分子機(jī)制。研究目的1.構(gòu)建miR-184基因過表達(dá)及干擾慢病毒載體;2.探究miR-184在NSCs中的目標(biāo)靶基因;3.研究miR-184對NSCs增殖分化的影響及其分子機(jī)制;4.研究miR-184與Notch信號通路在NSCs增殖分化時的相互作用機(jī)制。研究方法1.通過RT-PCR技術(shù)擴(kuò)增出miR-184基因和miR-184 shRNA基因,并分別克隆到pHBLV-U6-Scramble-ZsGreen質(zhì)粒載體中,構(gòu)建慢病毒表達(dá)載體。分離培養(yǎng)孕14天的胎鼠腦室下區(qū)(SVZ區(qū))的神經(jīng)干細(xì)胞并鑒定。2.實(shí)驗(yàn)細(xì)胞分為過表達(dá)對照組、miR-184過表達(dá)組、干擾對照組和miR-184干擾組,每組分別感染對應(yīng)的慢病毒,并用終濃度為3 μg/mL的嘌呤霉素,進(jìn)行抗性篩選,收集各組存活細(xì)胞繼續(xù)培養(yǎng)至3代。通過RT-qPCR實(shí)驗(yàn),進(jìn)行miR-184過表達(dá)驗(yàn)證。3.采用Brdu細(xì)胞滲入實(shí)驗(yàn),檢測各組細(xì)胞增殖情況,記錄各組Brdu+/DAPI+的比例。用神經(jīng)干細(xì)胞分化培養(yǎng)液培養(yǎng)各組NSCs,細(xì)胞免疫熒光法檢測NSCs分化為神經(jīng)元細(xì)胞的情況,記錄MAP2+/DAPI+比例。并檢測NeuN蛋白和NeuNmRNA表達(dá)水平,以及Notch通路相關(guān)蛋白Hes 1、Hes5及其mRNA表達(dá)水平。4.通過 targetScan、iRTarBase、miRanda 等軟件預(yù)測到 Numbl 作為 miR-184的潛在靶基因,經(jīng)Western blotting法及RT-qPCR法進(jìn)行NSCs胞內(nèi)靶基因驗(yàn)證,并通過雙熒光素酶報(bào)告基因系統(tǒng)進(jìn)行驗(yàn)證。研究結(jié)果1.成功構(gòu)建 pHBLV-U6-miR-184/miR-184-shRNA-ZsGreen 慢病毒表達(dá)載體。免疫熒光結(jié)果顯示分離培養(yǎng)的細(xì)胞90%呈Nestin和Sox2雙陽性。2.細(xì)胞成功感染慢病毒,抗性篩選培養(yǎng)后,細(xì)胞生長良好,RT-qPCR結(jié)果顯示,miR-184過表達(dá)組的miR-184相對表達(dá)量是過表達(dá)對照組的67.63±7.53倍,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。3.Brdu細(xì)胞滲入實(shí)驗(yàn)等實(shí)驗(yàn)結(jié)果顯示,與過表達(dá)對照組相比較,miR-184過表達(dá)組Brdu+/DAPI+比例是過表達(dá)對照組的1.47 ±0.05倍,MAP2+/DAPI+比例是過表達(dá)對照組的0.88±0.03倍,而與干擾對照組相比較,miR-184干擾組Brdu+/DAPI+比例是干擾對照組的0.84±0.03倍,MAP2+/DAPI+比例是干擾對照組的1.18±0.03倍。與過表達(dá)對照組比較,miR-184過表達(dá)組的NeuN蛋白及其mRNA相對表達(dá)量明顯降低,分別是過表達(dá)對照組的0.76±0.02倍、0.75±0.07倍,而與干擾對照組相比較,miR-184干擾組則明顯升高,分別是干擾對照組的1.22±0.01倍、1.54±0.05倍,以上差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。與各自對照組比較,miR-184過表達(dá)組Hes1、Hes5蛋白及其mRNA相對表達(dá)量明顯升高,miR-184干擾組則明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。4.通過iRTarBase和miRanda等軟件,預(yù)測到Numb1是miR-184潛在的靶基因。與過表達(dá)對照組相比較,miR-184過表達(dá)組的Numb1蛋白的相對表達(dá)量明顯降低,是過表達(dá)對照組的0.73±0.07倍。與干擾對照組相比較,miR-184干擾組Numb1蛋白相對表達(dá)量則升高,是干擾對照組的1.30±0.05倍。差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。此外,miR-184過表達(dá)組Numb1 mRNA的表達(dá)水平是過表達(dá)對照組的0.98±0.07倍,差異無統(tǒng)計(jì)學(xué)意義(t=0.593,P=0.613)。結(jié)果表明miR-184可抑制Numb1蛋白的翻譯過程,而不改變Numb1 mRNA的表達(dá)水平。而且雙熒光素酶實(shí)驗(yàn)結(jié)果進(jìn)一步表明Numb1是miR-184的一個靶基因。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R329.2
本文編號：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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R329.2
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