胰岛素样生长因子结合蛋白-4对神经干细胞增殖分化的影响

发布时间：2018-01-29 23:13

本文关键词： 神经干细胞 IGFBP-4 脂质体转染电转仪转染增殖分化　出处：《华北理工大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的本实验拟通过构建胰岛素样生长因子结合蛋白-4(IGFBP-4)高表达质粒及干扰质粒,并运用脂质体和电转仪转染两种方法转染质粒,通过对比转染效率选取转染效果更好的方法,用最终选择的较高效的转染方法将所构建质粒转染至神经干细胞内,建立IGFBP-4高表达/低表达细胞模型,继而观察IGFBP-4对神经干细胞增殖、分化的影响。方法首先从大鼠脑组织中提取总RNA,RT-PCR扩增IGFBP-4基因,TA克隆扩增PCR产物后将目的基因片段和p EGFP-N1连接,并对阳性克隆进行测序来构建IGFBP-4高表达质粒,IGFBP-4干扰质粒;体外培养神经干细胞,分别用脂质体转染和电转仪转染两种方法向神经干细胞内转染所构建质粒,通过对比转染效率,选取可获得更理想效果的方法,并探寻不同质粒的最优转染条件;根据最优转染条件,转染各种质粒,并运用western blot对各组细胞IGFBP-4含量进行测定;转染后48h使用荧光免疫细胞化学法计数Nestin、GFAP、MAP-Ⅱ阳性表达的细胞数,观察神经干细胞的分化方向;运用i CELLigence实时无标记细胞功能分析仪监测寻找对于E-plate L8板最适的种植密度,并对比在最适种植密度下同一时间点各组细胞曲线的高度。所有数据均采用SPSS17.0软件进行统计分析。结果1成功提取并扩增IGFBP-4基因,重组质粒pEGFP-N1-IGFBP4经酶切和序列分析鉴定与预期设计一致;2运用脂质体Lipofectamine LTX和电转仪CUY21EDITII两种转染方法均成功将质粒导入神经干细胞中,并在干细胞中有效表达,但两种方法转染效率差别明显,脂质体转染效率为30~40%,电转仪转染效率可达80%以上,电转仪转染效率明显高于脂质体转染;3选择电转染方法作为本实验导入质粒的方法,成功转染4组质粒,并获得每种质粒的最优电转染条件为:p EGFP-N1质粒为Pp V(Poration pulse Voltage)350V,Pd V(Driving pulse Voltage)20V,脉冲时间为10ms,循环次数为20次;IGFBP-4高表达质粒的最优条件:Pp V为400V,Pd V为75V,脉冲时间30ms,循环次数50次;IGFBP-4干扰质粒的对照质粒最优条件是:Pp V为400V,Pd V为60V,脉冲时间18ms,循环次数40次;IGFBP-4干扰质粒的最优条件:Pp V为400V,Pd V为35V,脉冲时间20ms,循环次数40次;4对转染成功48h的四组细胞进行western blot实验,发现转染IGFBP-4高表达质粒组细胞表达蛋白IGFBP-4含量明显增高,较对照组提高1倍左右,IGFBP-4干扰质粒组蛋白IGFBP-4含量明显降低,干扰效率达80%,说明成功构建IGFBP-4高/低表达细胞模型;5 i CELLigence实时无标记细胞功能分析仪监测不同种植密度下细胞的生长曲线,最适的种植密度为每孔5×104个细胞;6选取第100h对比各组曲线高度,IGFBP-4高表达组高于其对照组,IGFBP-4干扰质粒组低于其对照质粒组,IGFBP-4高表达组明显高于IGFBP-4干扰质粒组,差别有统计学意义;7免疫荧光观察各组细胞分化情况:IGFBP-4高表达对照组MAP-Ⅱ阳性细胞比例为27.2%,GFAP阳性细胞比例为32.3%;IGFBP-4高表达质粒组神经元细胞标志物MAP-Ⅱ阳性细胞比例为38.0%,胶质细胞标志物GFAP阳性细胞比例为92.0%;IGFBP-4干扰质粒对照组MAP-Ⅱ阳性细胞比例为21.7%,GFAP阳性细胞比例为86.1%;IGFBP-4干扰质粒组MAP-Ⅱ阳性细胞比例为24.3%,GFAP阳性细胞比例为89.6%。IGFBP-4高表达组神经元分化比例大于PEGFP-N1质粒组(P0.05),IGFBP-4干扰质粒组神经元分化比例较其对照组RNAi-negative control组明显降低(P0.05)。结论1 IGFBP-4高表达/低表达细胞模型构建成功;2 IGFBP-4促进了神经干细胞的增殖;3 IGFBP-4促进了神经干细胞向神经元方向的分化。
[Abstract]:The purpose of this experiment is to construct the insulin-like growth factor binding protein -4 (IGFBP-4) high expression plasmid and the plasmid, and the use of liposomes and electroporator transfection of two plasmids transfection method, method of selecting better transfection efficiency by comparing with the transfection method the final selection of the more efficient the constructed plasmid was transfected into neural stem cells, the establishment of IGFBP-4 high expression / low expression cell model, then observe the effect of IGFBP-4 on the proliferation of neural stem cells, in vitro. Firstly, total RNA was extracted from rat brain tissue, IGFBP-4 gene was amplified by RT-PCR, cloned TA PCR amplification products after the target gene fragment and P EGFP-N1 connection, and the positive clones sequencing to construct the high expression of IGFBP-4 plasmid, IGFBP-4 plasmid; neural stem cells cultured in vitro respectively by liposome transfection and electroporator transfection two methods to transfection of neural stem cells Through the comparison of the constructed plasmids, transfection efficiency, selection method can obtain better effect, and to explore the optimal transfection conditions of different plasmids; according to the optimal conditions of transfection, transfection plasmid and the use of Western blot on the content of IGFBP-4 cells were measured; 48h after transfection using fluorescence count immunocytochemical method Nestin, GFAP, cell number the positive expression of MAP- II, to observe the differentiation direction of neural stem cells; real-time ncell function analyzer to find the most suitable for monitoring E-plate L8 board using I CELLigence and planting density, the optimum planting density contrast in the same time the cells were the height of the curve. All the data were analyzed by SPSS17.0 software. Results 1 the successful extraction and amplification of IGFBP-4 gene, the recombinant plasmid pEGFP-N1-IGFBP4 by restriction enzyme digestion and sequence analysis was consistent with the expected design; 2 Lipofectamine by liposome. LTX and electroporator CUY21EDITII two transfection methods were successfully plasmid into neural stem cells, and effective expression in stem cells, but the transfection efficiency of two methods have obvious differences, the transfection efficiency of liposome 30~40%, electroporator transfection efficiency can reach more than 80%, electroporator transfection efficiency was significantly higher than that of liposome transfection 3; electric transfection method as the experimental method into the plasmid, plasmid successfully transfected into 4 groups, and obtain the optimal transfection conditions for each plasmid: P EGFP-N1 plasmid Pp V (Poration pulse Voltage) 350V Pd V (Driving pulse Voltage) 20V, the pulse time is 10ms, the number of cycles for 20 optimal conditions; the high expression of IGFBP-4 plasmid: Pp V 400V, Pd V 75V, pulse duration 30ms, 50 cycles; the optimal control conditions of plasmid IGFBP-4 interference plasmid is Pp V 400V, Pd V 60V, pulse duration 18ms, 40 cycles; IGFBP-4 interference The optimal conditions: Pp V plasmid 400V, Pd V 35V, pulse duration 20ms, 40 cycles; 4 to four groups of cells transfected successfully 48h Western blot experiment, found that cells transfected with IGFBP-4 expression plasmid group and high expression of IGFBP-4 protein content was significantly higher than the control group increased 1 times, IGFBP-4 interference plasmid protein content of IGFBP-4 was decreased and the interference efficiency was 80%. The successful construction of IGFBP-4 high / low expression cell model; 5 I CELLigence real-time growth curve labeled cell function analyzer in monitoring cells under different planting densities, the optimum planting density for each hole 5 * 104 cells in 6 groups were compared; choose the 100h curve high, high expression of IGFBP-4 group is higher than that of the control group, IGFBP-4 interference plasmid was lower than that of control plasmid group, high expression of IGFBP-4 group was significantly higher than that of IGFBP-4 plasmid group, the difference was statistically significant; 7 immunofluorescence to observe the fine Cell differentiation: high expression of IGFBP-4 positive cells proportion control group MAP- II was 27.2%, the proportion of GFAP positive cells was 32.3%; the high expression of IGFBP-4 plasmid group neuron cell marker positive cells were MAP- II 38%, glial cell marker positive cells of GFAP is 92%; IGFBP-4 interference plasmid control group MAP- II positive cells ratio was 21.7% for 86.1%, the proportion of GFAP positive cells; IGFBP-4 interference plasmid MAP- II positive cells accounted for 24.3%, the proportion of GFAP positive cells in 89.6%.IGFBP-4 high expression group neuron differentiation ratio greater than PEGFP-N1 plasmid group (P0.05), IGFBP-4 plasmid group neuronal differentiation ratio significantly compared to the control group RNAi-negative control group decreased (P0.05). The low expression cell model the successful construction of the high expression of IGFBP-4 IGFBP-4 2 / 1 conclusion; promote the proliferation of neural stem cells; 3 IGFBP-4 promotes neural stem cells into neurons Differentiation.

【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R741

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