| The clinical observation has shown that interferon-alpha (IFN- α ) is one of the most effective therapeutic agents for the malignancies of hemopoietic system and lymphoma. However, IFN- a can only induce about 70%~80% of the patients with chronic myeloid leukemia (CML) patients to get hematological remission. The molecular mechanism of IFN- a efficacy in the treatment of CML is still unclear. By comparing the reactivity of the two CML cell lines (KT-1/A3 and K562) toward IFN- a , the results approved that KT-1/A3 cells were more sensitive to IFN- a than that of K562 cells. And then we constructed a cDNA library of IFN- a -induced genes by means of suppression subtractive hybridization (SSH) from KT-1/A3 cells induced with IFN- a . It might be useful to analyze the function of the differentially expressed genes for the elucidation of the mechanism of IFN- a efficacy in the treatment of CML. Objectives:By comparing the reactivity of the two CML cell lines (KT-1/A3 and K562) toward IFN-α , we chose KT-1/A3 cells for studying the molecular mechanism of the suppression of Ph+ clone cells from CML by IFN- a . And then we constructed a subtracted cDNA library of IFN-α induced differentially expressed genes by means of SSH from KT-1/A3 cells induced with IFN-α . The identification and function studies of IFN- α induced genes will prompt the elucidation of the molecular mechanism of IFN- a in the treatment of CML.Methods:1. Comparing the sensitivity of the two CML cell lines (KT-1/A3 and K562) toward IFN- α . ①The effects of IFN- α in different concentrations (100, 500, 1000, 5000 and 10000 IU/ml) on growth ofthe two CML cell lines were detected by MTT assay , semisolid colony formation and trypan-blue staining for the cells in liquid culture. ①The cell apoptosis was examined by flowcytometry (FCM), fluorescence microscopy and gel electrophoretic analysis for DNA fragmentation at 48h after IFN- α (1000IU/ml) induction of both KT-1/A3 and K562 cell lines. #The expression levels of bcr-abl chimeric genes were analyzed by the relatively quantitative RT-PCR at 48h after cultivation of both KT-1/A3 and K562 cell lines with IFN- α in 1000IU/ml.2. On the base of the above experiments, we induced KT-1/A3 cells by IFN- α in 1000 IU/ml for 48 hours and parallel with KT-1/A3 control cells without IFN- α induction, then constructed a subtracted cDNA library of IFN- α induced differentially expressed genes by means of SSH from KT-1/A3 cells induced with IFN- a or without IFN-α induction. #1The mRNA was purified from total RNAs isolated from KT-1/A3 control cells and KT-1/A3 cells induced by IFN- α . The cDNAs were synthesized by reverse transcriptive reaction and disgested by Rsa I. The cDNAs from KT-1/A3 control cells.were used as driver and those from IFN- α induced cells were used as tester. Tester cDNAs were divided into two parts and ligated with different adaptors. After denatured, these two tester cDNAs hybridized with driver cDNAs respectively. Then the two reactions were merged, and further hybridized with excessive denatured driver cDNAs. So the genes expressed in both KT-1/A3 control cells and KT-1/A3 cells induced by IFN- α with the same level were subtracted, while the IFN- α -induced transcripts were accumulated. Suppression PCR was used (the primer was the adaptor sequence ) to amplify cDNAs of IFN- α induced genes. Those cDNAs were ligated with T vector and transformed into Ecoli DH5 a . The differentially expressed genes subtracted cDNA library of IFN-alpha -induced in KT-1/A3 cells was pooled. ② By using the blue-white screen and the Amp resistance screen, we chose the white single clones, prepared plasimids and identified the positive clones with EcoR I digestion. We acquired 22 differential expressed cDNA sequences by using EcoR I and Hae III to digest the PCR products ofthe positive clones.3. Iidentifying the differentially expressed genes. The 22 positive clones were screened by reverse Northern blotting analysis. The 1... |
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