The Experimental Study Of Acute Leukemia Cell Apoptosis Induced By Inhibition Of CREB Gene By RNA Interference

Leukemia is a cloned malignant disease by the hematopoietic stem cells. The cloningleukemia cells stay in the different stages of cell development due to lose furtherdifferentiation mature ability and stagnation.,The cells accumulating in bone marrow andother hematopoietic organizations make normal hematopoiesis inhibited. The acute leukemiahas become the most common malignant tumors in the children period. The treatment forleukemia include chemotherapy, the treatment of combining traditional Chinese and westernmedicine, the treatment of biological preparation, the treatment of bone marrow transplant tand so on. They are not ideal. For instance,radio and chemotherapies would inevitably resultin serious side effects.RNA interference(RNAi)technology,one of the novel gene therapies for disease diagnosis and treatment,can effectively hinder disease progression by identifying and interferingwith the pathogenic target genes.Recently it has drawn increasing attention in the treatmentof tumors. The research of CREB gene in lung cancer, liver cancer and other kinds of solidtumors become more and more, and the research in leukemia is also in further studies.Related experiments show if the amount of CREB is reduced. tumor cell the survival andrelated growth of tumor cell can be inhibited.This study adopts the technology of RNAi in the gene therapy, and takes the CREBgene as an therapy target, constructing RNAi plasmid for CREB gene. And this studyinduces the plasmid into acute leukemia cell successfully in vitro. this study tests apoptosisof acute leukemia cell in vitro by the method of RT-PCR, Western-blot, flow cell technology,MTT, etal.Further testing the suppression effect of tumor after interfering CREB gene byRNAi.Study Aim: suppressing CREB gene by RNAi technology in vitro, then, reach thetarget of suppressing acute leukemia cell.Study method:(1)According to the principle of RNAi screening, design the RNAi template for CREBgene in vitro, using restriction enzyme to cut the template and plasmid simultaneously. afterthat, using T4connection enzyme to connect the linear plasmid carrier and aim gene together,constructing RNAi plasmid for CREB gene，namely psilencer1.0--CREB-siRNA (2)Prepare feeling-state DH5α cells in vitro，then induce the reconstructed plasmid intothe cells，then extract the reconstructed plasmid and identify it. They are divided into fourgroups: psilencer1.0--CREB-siRNA; psilencer1.0--control-siRNA; psilencer1.0; Sterilewater. We preparation SDS-polyacrylamide gel electrophoresis to finish western-blotingexperiment.Then we extract and quantify RNA in the cell,and get cDNA to take theretrovirus reaction. We inspect the apoptosis of the acute leukemia cell by the flowcytometric technigy, detect the survival rate of living cells by MTT method to judge t theinhibition effect of CREB by the RNAi.The experimental results:(1) The relevant appraisal of enzymes cutting test out of the psilencer1.0--siRNAplasmid CREB to be built successfully.(2) In vitro We through Western-blot technical inspection protein of psilencer1.0-CREB-siRNA plasmid is significantly reduced by72.1%.(3) In vitro We through RT-PCR technical inspection gene of psilencer1.0-CREB-siRNA plasmid is significantly reduced by71.7%.(4) We take the MTT method to detect cell proliferation, The cellular growth ofpsilencer1.0--siRNA plasmid is inhibited. Inhibition rate of48h is21.9%, and inhibition rateof72h is22.6%.(5) The flow cytometer test suggests that cell cycle was blocked by recombinantplasmid at the G0/G1phase,with hypodiploid peak present before normal diploid peak.Theapoptosis rate was high.The experimental results: We successfully constructed U937cell lines of psilencer1.0-U6-siRNA plasmid, both the genes and protein level is decreased obviously. In order toverify the molecular mechanism of the pro-apoptosis and proliferation inhibition effect ofrecombinant plasmid,MTT test and cell cycle test by flow cytometer were performed. Thecell became apoptosis obviously, RNAi molecules can be as a treatment molecules for acuteleukemia.