| IntroductionWT1, originally isolated from Wilms' tumor cells, is at chromosome 11 pi3, spans about 50kb and comprises ten exons as well as mRNAs of approximately 3kb. WT1 proteins - DNA binding factors, have molecular masses of 52 - 54kDa. In recent years, researchers explored its role on hematopoiesis. They have found that embryo cells lacking WT1 show deficits in hematopoietic stem cell function. WT1 can regulate the expression of the CSF-1, IGF-IR, bcl-2, c-myc and retinoic acid receptor genes. So it regulates the hematopoiesis in this way. In leukemia cells, WT1 has abnormal functions and expressions : an aberrant overexpression of wild - type WT1 in leukemia cells; an inverse correlation between WT1 expression levels and prognosis; an increase in WT1 expression levels at relapse; inhibition of differentiation and induction of proliferation myeloid progenitor cells in response to G - CSF stimulation; Some leukemias have mutations in WT1 gene, which produce truncated proteins with ruinated zinc - finger domain. Due to the absence of zinc -finger domain, WT1 protein can not regular the target genes and control the development , the proliferation and the division of the cells. So did its negative control on itself. These phenomenon show that WT1 gene is a tumor gene during the development of the leukemia.WT1 gene antisense oligonucleotides complement with original transcript part of WT1 mRNA, which contain 18 nucleotides.The object of this experiment is to inhibit the expression of WT1 gene by antisense oligonucleotides , to observe the changes of morphology and proliferation behavior of leukemia cells, to research the role of the WT1 in the genesis and development of leukemia and discuss the application of antisense oligonucleotides on bone marrow depletion and antisense therapy.MaterialsMain materials and equipmentFicoll lymphocyte separative liquid , Trizol, chloroform, dNTP, RNA AMV , RPMI1640 , Taq DNA polymerase are provided from Infectious Disease Experiment Lab. Primers and oligonucleotides are purchased from biological company. CO2 gas incubator, -152 C medical freezer, PCR equipment.MethodsWT1 gene assay and cell cultureLeukemia cells , expressing the WT1 gene , drawn out of patients' bone marrow , were cultured in RPM1 1640 which contain 10% heat-inacted fetal bovine serum, 50ug/ml streptomycin and 100IU/ ml penicillin and incubated at 37 C in 5% CO2 atmosphere. The cells in the logarithmic growth phase are used in experiment.Antisense oligonucleotides preparationDesign the antisense oligonucleotide to complement with the original sites for translation. The sequence is followed: sense strand:5' CAGCAAATGGGCTCCGAC3'; antisense strand:5'GTCGGAGC-CCATTTGCCTG3', which were modified with sulfration and purified with HPLC.Cells treatment with antisense oligonucelotideLeukemia cells, are divided into three groups and separately cultured with antisense oligonucleotides, sense oligonucleotides and RP-MI1640. We add the reagents with 40ug/mL per 24 hours until 96 hours.Assays for WT1 gene expressionCollecting cells of each group after 96 hours and detecting the expression of WT1 gene by RT - PCR .Assays for the changes of morphologyDyeing the cells before and after experiment observing the morphological changes under the microscope.Assays for the proliferation of cellsCounting the cells per 24hours and computing the relative survival percent : amount of experiment group cells/control group cells x 100%.Result1. The expression of WT1 geneCollecting each group cells after treatment with antisense oligonu-cleotids for 96 hours, we detected the WT gene by RT - PCR. The result is that WT1cDNA in AS group was disappeared, while that in both S and control groups could still be detected. We can conclude that this antisense oligonucleotide can inhibit the expression of WT1 gene effectively.2. Assays for the morphological changesThe cells after treatment with antisense oligonucleotide were dead a lot. This phenomena shows tha... |
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