| Objectives The study aims to construct IGF-binding protein-4(IGFBP-4) highexpression and interference plasmid. Comparing the transfection efficiency between using liposome and electroporation apparatus to transfect plasmids into cells.Then the method that gets high transfection efficiency is chosen as the final choice to transfect the plasmids into neural stem cells to establish IGFBP-4 high / low expression cell model, and observe the effect of IGFBP-4 on the proliferation, differentiation of NSCs.Methods Firstly, total RNA was extracted from rat brain tissue and IGFBP-4 gene was amplified by RT-PCR and TA cloning, then the target gene fragment was connected with p EGFP-N1 Plasmid. Positive clones were sequenced to construct the IGFBP-4 overexpression plasmid and IGFBP-4 interference plasmid. Neural stem cells were cultured in vitro,then liposome and electroporation apparatus were used to transfect plasmids into cells. By comparing the transfection efficiency,a more ideal method was chosen to explore the optimal transfection conditions of different plasmids. According to the optimal transfection conditions, all kinds of plasmids were transfected to measure the content of IGFBP-4 in each group cells by using western-bloting; the NSCs with Nestin/GFAP/MAP-2 positive expresstion were counted at 48 h after transfection by fluorescence immunocytochemistry, and the differentiation direction of neural stem cells was observed;Using real-time label-free i CELLigence cell function analyzer to explore the most suitable planting density for E-plate L8 board by contrasting the height of the curve of each group at any time point under the optimum planting density.All data are statistically analyzed by using SPSS17.0 software.Results 1 Extracting and amplifying IGFBP-4 gene successfully, the recombinant plasmid p EGFP-N1-IGFBP4 tested by enzyme digestion and sequence analysis are consistent with the expected design. 2 Lipofectamine LTX and electroporation apparatus CUY21 EDIT Ⅱ can transfect plasmids into NSCs and effective expression successfully.However, the difference is significant, Liposome transfection efficiency is 30~40%, while the electroporation device is up to 80%. 3 The transfection efficiency of electroporation was significantly higher than Liposome transfection. We chose the electroporation as the main method and transfected every kind of plasmid into cells successfully.The best conditions for every plasmid were as following: p EGFP-N1 plasmid( 350 V Pp V, 20 V Pd V, 10 ms pulse time,20 cycles);IGFBP-4 over-expression plasmid( 400 V Pp V,75V Pd V,30 ms pulse time,50 cycles);RNAi-negative control plasmid(400V Pp V, 60 V Pd V,18 ms pulse time,40 cycles);IGFBP4-RNAi plasmid(400V Pp V, 35 V Pd V,20 ms pulse time, 40 cycles). 4 The result of Western blot experiment on four groups of cells 48 h after transfection were as following:the protein expression of IGFBP-4 in the IGFBP-4 highexpression group was significantly higher than those in the control group, increased about one times, while IGFBP4-RNAi plasmid group decreased significantly, the interference efficiency was up to 80%,indicating the successful construction of IGFBP-4 high/low expression cell models. 5 The growth curves of G10 cells under different density which were got from i CELLigence system and the suitable density was 5×104 in every hole. 6Comparing the curve at the 100 thhour among these groups,the group with IGFBP-4 highexpression was higher than the group with p EGFP-N1 plasmid,the group with IGFBPRNAi plasmidwas lower than the group with RNAi-negative control plasmid,the group with IGFBP-4 high-expression was higher than the group with IGFBP-4-RNAi plasmid.The difference was statistically significan. 7 The differentiation of cells in every group observed under fluorescence microscope:the percentage of neurons was 27.2% in the group with p EGFP-N1 plasmid,the percentage of glial cells is 32.3%;the percentage of neurons was 38.0% in the group with IGFBP-4 high-expression,the percentage of glial cells was 92.0%; the percentage of neurons was 21.7% in the group with RNAi-negative control plasmid,the percentage of glial cells was 86.1%;the percentage of neurons was24.3% in the group with IGFBP4-RNAi plasmid,the percentage of glial cells was 89.6%;the percentage of neurons in the group with IGFBP-4 high-expression was higher than the group with p EGFP-N1 plasmid(P<0.05); the group with IGFBP-RNAi plasmid was lower than RNAi-negative control plasmid(P<0.05).Conclusions 1 IGFBP-4 high/low expression cell modesl are successfully constructed; 2IGFBP-4 promotes the proliferation of NSCs; 3 IGFBP-4 promotes the percentage of neurons which differentiated from NSCs. |
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