The Experimental Research On The Construction Of RNAi Eukaryotic Expression Vector And The Interference Effect After Transfect To Leukemia Cell Lines

Objective:The p53 is one of the best-known anti-oncogenes. The new discovered protein ASPP(apoptosis-stimulating protein of p53) can specific regulated the tumour suppress of p53. iASPP can interfere the apoptosis of p53 through binding the p53 ,so it is the important inhibit protein of p53. To understand the relationship between ASPP and the leukemia cells ,we detected the mRNA expression levels of ASPP members in three kinds of leukemia cell lines by RT-PCR.We construct the RNAi eukaryotic expression vector of long iASPP gene and transfect it into leukemia cell lines by liposome. Then we observe the interference effect of iaspp by RT-PCR,Western Blot and detect the cell apoptosis by flow cytometry.We explore the ability of regulate p53 by RNAi and try to find the new strategy to improve the tumor threapy, and then find a means for further experiments.Methods:1. Leukemia cell lines HL-60,Jurkat,Molt-4 were grown in RPMI medium 1640 supplement with 10% fetal calf serum and 100U/ml benzylpenicillin and 100 U/ml streptomycin sulfate in a humidified atmosphere containing 5% of CO2 at 37oC.2. mRNA expression levels of ASPP gene family were measured by RT-PCR.3. The specific small interference RNA sequence was designed according to the genebank sequence of iASPP, the template sequence was cloned into the PGCsilencerTM H1/Neo/GFP plasmid and then analyze the sequence.4. The recombinant plasmid was transfected into leukemia cell lines by liposome, the iASPP expression was analyzed by RT-PCR and Western Blot .The cell apoptosis was detected by FCM.Results:1. ASPP gene family mRNA was expression in all leukemia cell lines, but the...