The Role Of NM23 In Hepatocyte Proliferation

The first partThe overexpression vector and interference vector of NM23-E2 construction, expansion, identificationObjective:Build, amplification, identification, screening the overexpression vector and interference vector of NM23-E2 for follow-up study to lay the theoretical foundation.Methods:1.NM23-E2 and HPV-16 E6, E7 slow virus, Building slow virus vector and sequencing;2.Recombinant lentivirus plasmid extraction;3.Building NM23-E2 lentivirus transfection HEK293T cells;4.The detection of virus drops;5.Restructuring NM23-E2 slow virus and Infecting T293 cells and screening;6.Application of RT-PCR detected NM23-E2 mRNA7. Western blot detection NM23-E2 protein.Results:Successful building the overexpression vector and interference vector of NM23-E2, sequencing validate and correct;Through transfection THEK293T cells, determination of virus drops degree;Q-PCR, Western blot tests from shRNA1, shRNA2, shRNA3, shRNA4 four effectiveness sequence of screened,Filtering out interference effect best shRNA1 as interference carrier.Conclusion:Successful build NM23-E2 slow virus carrier, sequencing, correctly selected carrier interference effect is the best interference and high expression of expression vector.The second part The function of NM23-E2 in liver cell regenerationObjective:Researching NM23-E2 gene playing the role in the process of BRL-3A cell proliferationMethods:Experimental points five groups:1.the control group (control), without any processing,2.express group (Exp NM23-E2), transfection NM23-E2 expression vector;3.the overexpression of NC group (Exp NC), transfection NM23-E2 negative control expression vector,4. interference of NC (shRNA NC), transfection NM23-E2 interference negative control carrier;5. the interference group (shRNA1), transfection NM23-E2 carrier interference.Will wrapped the above five kinds of carrier respectively transfection rats BRL-3A cell, the fifth day after transfection application Q-PCR detection BRL-3A cell NM23-E2, PCNA, Cyclin D1 mRNA expression, using Western blot detection BRL-3A cell NM23-E2, PCNA, Cyclin D1 protein expression, application CCK8 testing BRL-3A cell transfection cell proliferation of 1 to 5 days later.Results:PCR:Detecting mRNA expression of BRL-3 A cell NM23-E2, PCNA, Cyclin D1;NM23-E2 a set of three kinds of protein overexpression of mRNA expression quantity are higher than blank control group (P< 0.05), shRNA interference group, all three kinds of protein mRNA expression of the quantity is lower than the blank control group (P< 0.05), the difference was statistically significant.Western-blot:Detecting protein expression of BRL-3 A cell, NM23-E2、PCNA and CyclinDl protein:NM23-E2 had three kinds of protein expression quantity expression group were higher than in blank control group (P< 0.05), three kinds of protein expression quantity shRNA interference group were lower than in blank control group (P< 0.05), the difference was statistically significant.CCK8:Detecting BRL-3 A cell NM23-E2, PCNA, Cyclin D1、mRNA cell proliferation, cell proliferation number of NM23-E2 over expression group was obviously higher than that of blank control group and NC group cells (P< 0.05), the number of cell proliferation shRNA interference group was obviously lower blank control group and NC group cells (P<0.05), the difference was statistically significant.Conclusion:NM23-E2 may play a role in the process of BRL-3 A cell proliferation...