干扰及过表达miR-31对脊髓源神经干细胞分化的影响

发布时间：2018-05-06 22:09

本文选题：脊髓损伤 + 神经干细胞　；参考：《山西医科大学》2015年硕士论文

【摘要】：目的:1.使用Alexa Fluor R Red检测RNAi MAX对神经干细胞的转染效率,确定最终的转染效率最高的时间。2.观察mi R-31对神经干细胞向运动神经元分化时所起作用,研究mi R-31和神经干细胞之间的相互关系。方法:第一部分:RNAi MAX对神经干细胞的影响及转染效率的测定首先从胎鼠体内获得脊髓源神经干细胞,体外分离培养并鉴定。当干细胞传至3代后,接种于铺设多聚赖氨酸处理过盖玻片的六孔板中,分为不加RNAi MAX组和加RNAi MAX两组,通过0d、1d、2d和3d的连续观察,研究RNAi MAX对神经干细胞的影响作用。并继续将细胞分为1d、2d和3d三组,转染Alexa Fluor R Red观察RNAi MAX对神经干细胞的转染效率。当达到相应的时间点时,取出细胞并用多聚甲醛固定,之后将细胞位于荧光显微镜下观察,计算发出红色荧光细胞和总细胞数之比确定RNAi MAX对神经干细胞的转染效率。第二部分:mi R-31对神经干细胞的诱导作用将细胞分为干扰组、干扰对照组、过表达组、过表达对照组和对照组,转染相应试剂后观察细胞的形态学变化,并通过Ch AT的免疫细胞化学染色观察胆碱能神经元的分化情况。提取RNA后检测mi R-31的诱导是否成功,并反转录为c DNA后通过荧光定量PCR检测下游基因在mi R-31的表达变化后的变化情况。通过以上分析mi R-31在神经干细胞干性维持方面的作用。结果:第一部分:当RNAi MAX加入到神经干细胞后,细胞开始死亡。通过在时间节点镜下观察可知,在第一天时转染效率是20%-30%,第二天时转染效率是40%-55%,当达到第三天时,转染效率为60%-75%。说明Alexa Fluor R Red会随着时间的推移增加转染的细胞数,提高细胞的转染效率。第二部分:转染mi R-31的mimic和inhibitor后,细胞的形态和分子层面都发生了变化。形态学方面可以看出,转染mi R-31的抑制剂后,细胞的形态更接近于运动神经元,细胞呈长梭形,在免疫细胞化学染色中可以看出Ch AT的表达升高,细胞荧光呈强阳性;而在转染mi R-31过表达试剂后,细胞贴壁后形态变化不明显,突起伸出不多,Ch AT的表达明显降低,荧光强度明显弱于抑制组。micro RNA的反转录产物荧光定量PCR结果显示干扰组的表达为0.867,干扰对照组的表达为2.406,过表达组为1.022×106,过表达对照组为1.506×105,说明细胞转染成功。而下游基因也有明显变化。在干扰组中表达明显升高的有hb9和stmn1,表达降低的有Nestin、lats2、Rhobtb和SATB2。过表达组明显升高的有Nestin、hb9、lats2和SATB2,表达降低的基因有Rhobtb。结论:1.RNAi MAX对神经干性毒性较大,会造成大量的细胞死亡,在实验时需加大细胞的用量。通过转染效率的测定,RNAi MAX在第三天的转染效率最高。2.mi R-31高表达对神经干细胞有很好的干性维持作用,抑制时神经干细胞向运动神经元的分化增加。
[Abstract]:Purpose 1. Alexa Fluor R Red was used to detect the transfection efficiency of neural stem cells (NSCs) by RNAi MAX. To observe the effect of mi R-31 on the differentiation of neural stem cells into motor neurons, and to study the relationship between mi R-31 and neural stem cells. Methods: in the first part, the effect of MAX on neural stem cells and its transfection efficiency were measured. Firstly, spinal cord derived neural stem cells were obtained from fetal mice and isolated and identified in vitro. When the stem cells were transferred to the third passage, the cells were inoculated into the six hole plates treated with poly-lysine and divided into two groups: no RNAi MAX group and RNAi MAX group. The effect of RNAi MAX on neural stem cells was studied by observing the effects of RNAi MAX on neural stem cells for 2 days and 3 days. The cells were divided into three groups: 1 d, 2 d and 3 d. The transfection efficiency of RNAi MAX on neural stem cells was observed by Alexa Fluor R Red. When the cells reached the corresponding time point, the cells were fixed with paraformaldehyde, then the cells were observed under fluorescence microscope, and the ratio of red fluorescent cells to total cells was calculated to determine the transfection efficiency of RNAi MAX to neural stem cells. The second part: the inductive effect of: mi R-31 on neural stem cells: interference group, interference control group, overexpression group, overexpression control group and control group. The morphological changes of the cells were observed after transfection of corresponding reagents. The differentiation of cholinergic neurons was observed by immunocytochemical staining of chat. After extraction of RNA, the induction of miR-31 was detected, and the change of downstream gene expression was detected by fluorescence quantitative PCR after reverse transcription into c DNA. The role of mi R-31 in dry maintenance of neural stem cells was analyzed. Results: the first part: when RNAi MAX was added to neural stem cells, the cells began to die. The transfection efficiency was 20-30on the first day, 40-55on the second day, and 60-75on the third day. The results showed that Alexa Fluor R Red could increase the number of transfected cells and increase the transfection efficiency with time. The second part: after transfection of mimic and inhibitor of miR-31, the morphology and molecular level of the cells were changed. Morphological analysis showed that after transfection with the inhibitor of miR-31, the morphology of the cells was more similar to that of the motoneurons and the cells were fusiform. In the immunocytochemical staining, the expression of chat was increased and the fluorescence of the cells was strongly positive. However, after transfection of mi R-31 overexpression reagent, the morphological changes of the cells were not obvious, and the expression of Chat, which was not protruded and protruded, was significantly decreased after the transfection of mi R-31 overexpression reagent. The fluorescence intensity was significantly weaker than that in the inhibition group. The fluorescence quantitative PCR showed that the expression of the reverse transcription product was 0.867 in the interference group, 2.406 in the interference control group, 1.022 脳 10 6 in the overexpression group and 1.506 脳 10 5 in the over-expression control group, which indicated that the cell transfection was successful. And the downstream gene also has the obvious change. In the interference group, the expression of hb9 and stmn1 increased significantly, while the expression of nestinnlats2rhobtb and SATB2 decreased. In the overexpression group, Nestinhb9 lats2 and SATB2 were significantly increased, and Rhobtb was significantly decreased in the overexpression group. Conclusion: 1. RNAi MAX is more toxic to the nerve and can cause a large number of cell death, so the amount of cells should be increased in the experiment. The transfection efficiency of RNAi MAX was the highest on the third day. The high expression of 2.mi R-31 had a good dry maintenance effect on neural stem cells, and the differentiation of neural stem cells into motor neurons increased during inhibition.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.2

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