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<p><b>OBJECTIVE</b>To investigate the possibility of multi-unit ribozymes to purge bone marrow of chronic myelocytic leukemia (CML), its in vitro cleavage ability and the reversal effect on CML cell's malignant phenotype.</p><p><b>METHODS</b>As bcr-abl fusion gene plays an important role in CML pathology, three single-unit ribozymes were designed and synthesized in 44 base pairs near the fusion point, two enzyme cleavage sites on bcr gene and one on abl gene. Multi-unit ribozymes' in vitro transcription and retroviral vector through gene recombination were constructed. Then, its in vitro cleavage ability was tested and the retroviral vector was transfected into K562 cell. Through MTT assay, the incorporation rate of (3)H-TdR, RT-PCR, Southern and Northern blot hybridization, flow cytometry, transmission and scanning electron microscopy were used to study the effect of multi-unit ribozymes on CML cell proliferation, cell structure, cell cycle and the induction of apoptosis.</p><p><b>RESULTS</b>Multi-unit ribozymes had in vitro cleavage efficiency of 70.8%. After the transfection of multi-unit ribozymes retroviral vector into K562 cell, cell proliferation and DNA synthesis were greatly reduced with an inhibition rate of about 50% after 96 hours of transfection. Multi-unit ribozymes could cleave K562 cell's RNA with a reduction rate about one 1 000 th of the original. By flow cytometry (FCM), 18.4% cells underwent apoptosis after 72 hours transfection with most of the cells blocked in the G phase. Here, the ratio in S phase was lowered by 41.9%. Under transmission and scanning electron microscope, compaction of nuclear chromation and apoptosis bodies were observed in the transfected cells.</p><p><b>CONCLUSION</b>Multi-unit ribozymes possess high cleavage ability in vitro. The ribozymes, whose retroviral vector being transfected into CML cell, are able to express a lasting ability to cleave the fusion gene, induce apoptosis, reduce cell proliferation, revert the malignant phenotype. It is possible to make use of multi-unit ribozymes to purge CML bone marrow. Therefore, multi-unit ribozymes may very well be valuable in the gene therapy of CML.</p>
Subject(s)
Humans , Apoptosis , Cell Division , Fusion Proteins, bcr-abl , Genetics , Metabolism , K562 Cells , RNA, Catalytic , Metabolism , PharmacologyABSTRACT
Although adhesion molecules have long been recognized to be differentially expressed on naive and effector/memory T cells,it has recently been found that a number of chemokine receptors are also differentially expressed on T cells,depending on their Ag experience and type of polarization. Recent data suggests that chemokines and their receptors are essential elements that regulate the positioning of T cells and their partners for priming and T helper 1 (Th1)- or Th2-mediated responses,therefore,are probably the most promising targets for treating immune diseases.
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Objective:To isolate and establish long term T cell clones derived from bone marrow(BM) of a patient with severe aplastic anemia(sAA).Methods:CD34 + cells and CD3 +T cells were enriched from BM aspirates,respectively by positive and negative immunomagnetic sorting protocol.BM of exicised rib of a non hematological patient was used as control. Irradiated CD34 + cells together with T cells were subject to one way autologous mixed lymphocyte culture(AMLC).Viable T cells surviving from AMLC were limit diluted and single cell was plated into microtitier wells along with feeder cells.Then all wells were screened for the growing cell wells and they were transferred into new wells to be expanded and maintained.Each clones obtained were primarily assessed CD4/CD8 phenotype and their homogeneity by immunocytochemical analysis.Results:The purity of sorted CD34 + and T cells was more than 90%. On AMLC,a total of 4?10 3 cells were viable with sAA,but 65 cells viable with control.Upon single cell seeding it 11 growing cell wells were found for sAA,and no one growing cells for control.The actual plating rate(2.1%) was within Poisson theoretic value range (
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PURPOSE To investigate the effects of bcr3/abl2 anisense oligodeoxynucleotide (ASO) on chronic myeloid leukemia (CML). METHODS The effects of bcr3/abl2 ASO in 15 CML patients were evaluated by morphological observation, flow cytometer (FCM) techniques and DNA fragment measurement. RESULTS bcr3/abl2 ASO can induce apoptosis in CML. CONCLUSION Antisense techniques may provide a new way to cure CML.