| Objective:1. Transfection efficiency using Alexa Fluor R Red to detect RNAi MAX on neural stem cells, determined finally the highest transfection efficiency of time.2. To observe the effect of mi R-31 on neural stem cells into motor neuron differentiation role relationship between cells, the study of mi R-31 and neural stem. Methods:Part one: The effect of RNAi MAX on neural stem cells and transfection efficiencyWe first obtain the spinal cord derived neural stem cells from fetal rat in vivo, in vitro and identification. When the stem cells at passage 3, inoculated in the laying of polylysine treated 6-well plate glass, divided into RNAi MAX group and RNAi MAX plus plus two groups, by continuous observation of 0d, 1D, 2D and 3D, the study of effect of RNAi MAX on neural stem cell function. And continue the cells were divided into 1D, 2D and 3D three group, Alexa Fluor R Red to observe the transfection efficiency of transfection of RNAi MAX on neural stem cells. When reaching the corresponding time point, take out the cell and fixed with paraformaldehyde, after the cell located under the fluorescence microscope, determining the calculation issued RNAi MAX on neural stem cell transfection efficiency of red fluorescent cells and total cell number ratio.Part two: Mi R-31 guidance effect on neural stem cellsThe cells were divided into interference group, control group, group interference over expression, over expression of the control group and the control group, the change of cell morphology was observed after transfection of the corresponding reagent, and by immunocytochemical Ch AT staining to observe the differentiation of cholinergic neurons. After extracting RNA detection success induced by mi R-31, and the reverse transcription through changes in fluorescence quantitative PCR in detection of downstream genes in the expression change of mi R-31 c DNA after after. Through the above analysis of mi R-31 in neural stem cell stamens maintenance role. Research Results:Part oneWhen RNAi MAX was added to the neural stem cells, cells begin to die. On the first day of transfection efficiency is 20%-30%, second days of transfection efficiency is 40%-55%, when up to third days, the transfection efficiency of 60%-75%. Description Alexa Fluor R Red with the passage of time will increase the number of transfected cells, increase cell transfection efficiency.Part twoTransfection of mi R-31 mimic and inhibitor, morphological and molecular level cells have changed. Morphology can be seen, inhibitors after transfection with the mi R-31, the morphology of the cells was more close to the motor neurons, cells were long fusiform, in immunocytochemical staining can be seen in the increased Ch AT expression, cell fluorescence was strongly positive; while overexpression of reagents in mi R-31 after transfection, cells adherent morphology did not change significantly, projection out of not much, the expression of Ch AT was decreased, the fluorescence intensity was weaker than the inhibition of group. Micro RNA reverse transcription products of fluorescent quantitative PCR showed that the expression of interference group was 0.867, the expression of interference of control group was 2.406, over expression group was 1.022 ×106, over expression of control group is 1.506×105, shows cells transfected with success. And downstream genes have changed markedly. In the interference group significantly increased the expression of the Hb9 and STMN1, have reduced expression of Nestin, LATS2, Rhobtb and SATB2. Over expression group was significantly increased with Nestin, Hb9, LATS2 and SATB2, reduced expression of gene Rhobtb. Conclusion:1. RNAi MAX on the larger neural dry toxicity, can cause a large number of cell death in the experiment, the need to increase the amount of cells. Through the determination of transfection efficiency, RNAi MAX on the third day of the highest transfection efficiency.2. The high expression of mi R-31 have a good dry maintenance effect on neural stem cells, neural stem cells to inhibit when increased moto neuron differentiation. |
PDF Full Text Request