| Objective:By reviewing our preliminary research results, we found that as the expression of nucleostemin of p53 null HL-60 leukemic cells down-regulated,the expression of signal molecules of p38 MAPK pathway were obviously increased. This research was mainly to detect the changes of some important proteins in p38 MAPK signal pathway. Based on this research,we may make it clear the relationship between NS and p38 MAPK signal pathway, and explore the possible reasons for p38 MAPK pathway after NS was down-regulated,which make the foundation on studying details in mechanisms of p53-independent signal pathways of NS. Methods:(1) As our research team has already built for NS gene of recombinant lentivirus vectors(NS- RNAi- GV248) and negative vectors, for this research,we just purchase the lentivirus vectors directly from the company.(2) Using the most proper level of lentivirus vectors(NS-RNAi-GV248) to transfect into HL-60 cells,and its aim to down-regulate the expression of NS in the cells.After that,we observe the morphology of the HL-60 cells and the efficiency of transfection by inversion fluorescence microscope;using western blot technique to test the NS proteins in HL-60 cells.(3) Using western blot technique to detect the expression of p38 MAPK pathway related protein in blank control groups,negative control groups and experimental groups HL-60 cells,namely the ratio of p-p38MAPK/p38 MAPK.(4) The gray values of protein bands were analyzed with Image J image processing software. The datas were analyzed by SPSS 17.0 statistical software and were showed in the form with x± s,one-way ANOA methods was used to compare qucntitative datas,to further paried-comparisions using Bonferroni,α’=0.0167 was considered as calibration inspection level. Results:(1) The most proper MOI level of HL-60 cells is 80.Using this condition,the transfection efficiency of lentiviral vectors can reach more than 80%,and the expression of NS in experimental groups is lower than them in other groups.(2) Western Blot shows that the ration of and p-p38/p38 in experimental groups(0.444±0.008)is higher than them in negatve groups and blank groups[(0.286±0.016)and(0.261±0.019)](P<0.05).It refresh that p-p38 MAPK proteins increase with the NS proteins down-regulation.Conclusion:(1) The changes of signal molecules of p38 MAPK pathway,and the proteins of p-p38 MAPK was negatively correlated with NS down-regulation.(2) The procedure of NS p53-independent may be related with the key proteins of p38 MAPK pathway. |
