The Screening Of Transcription Factors Promoting Axonal Elongation In Mice

The limited regenerative ability of injured axon, which leads to an inefficient restruction of neural circuit, is a primary obstacle for functional recovery after nervous system injury. So far, most therapeutic strategies for neural regeneration mainly focus on three aspects: improving the local micro-environment in order to reduce adverse factors for axonal regeneration, transplantation of neural stem cells to replace the lost neurons and reconstrue the neural circuit, and stimulating the intrinsic regenerative ability of neuron. Among what have been mentioned above, to enhance the endogenous regenerative ability of neuron is essential for promoting injured nervous system repairation. Transcription factors(TFs), a kind of DNA-binding proteins that recurite to regulatory areas of genes to regulate the process of transcription in cis or trans ways, play important roles in determining genes function. In this study, dozens of TFs associated with axonal growth were screened by molecular and cellular biological techniques. TFs were transfected into PC12 cells and neurons in singly and multiply, respectively. Cell processed in different groups were observed and calculated. We attempt to find the individual or combinated TFs that can promote the elongation of cell processes in most efficiently.First, we chosed 38 TFs as candidate genes which have been reported to have certain roles in axonal generation by literature retrieval. Then, cloned the coding DNA sequences(CDS), sequencing and connected them into the modified eukaryotic expression vector, respectively. The expression vectors of 38 TFs were transfected into PC12 cells with liposome, respectively. The length of cell process was calculated. The results showed that all transfected TFs promoted the elongation of processes obviously with different levels. Among those transfected TFs, 10 TFs such as CREB, KLF4, p50, STAT3, Hb9 Batf, Sox10, SnoN, Lmx1 a and Lhx3 had the most ability to promot the length of cell process significantly, whereas others, such as ATF2, ATF3, E47 and Oct4, displayed limited promotion effects. The 10 TFs were as the candidates which were selected to give most promoting to elongation of processes in PC12 cells. However, before that, we have to answer two questions: the strategy of selection and whether is there such a combination of TFs is best for processes elongation? We chose 4 TFs(CREB, p50, STAT3 and KLF4) to test the elongation effects of combined transfection with the N-1 strategy. The combinations of TFs we designed were listed below: CREB+KLF4+p50, CREB+KLF4+STAT3, CREB+p50+STAT3, KLF4+p50+STAT3 to decide which TF was the most important for process elongation. The results showed that CREB and STAT3 were the most important for process elongation among the 4 TFs. The results suggest that our N-1 strategy is suitable to select the TFs and there is such a combination of TFs is best for processes elongation.Neural axonal growth ability can be enhanced by regulating neural genetic expression level. Our study not only establishes a theoretical foundation for study of neural injury and regeneration, but also provides a guidance for clinical treatment.