ABSTRACT
This study was purposed to construct a vector containing human suppressor gene p53 and p16, and to investigate their expression and effect on K562 and HL-60 cells. pBudCE4.1-53-16 is a vector designed for simultaneous expression of human suppressor gene p53 and p16 in mammalian cell line. After transfection into K562 cells with lipofectamine(TM) 2000, the expression of p53 and p16 genes was detected by Western blot and immunocytochemical method. The growth curve, apoptosis, cell cycle were assayed by CCK-8 and flow cytometry. The results showed that the recombinant plasmid pBudCE4.1-53-16 was constructed successfully and were verified by PCR and restriction analysis. The expression of P53 and P16 protein could be detected after transfection into leukemia cells (K562 and HL-60) for 48 hours. As compared with control group, the cell proliferation in experimental group was inhibited, the cells were arrested in G0 phase and apoptotic cells increased (p<0.001). It is concluded that the recombinant plasmid pBudCE4.1-53-16 has been established. p16 and p53 in the recombinant plasmid pBudCE4.1-53-16 synchronously express in leukemic cells after transfection in vitro for 2 days and results in reduced proliferation, G0 arrest and apoptosis increase.
Subject(s)
Humans , Apoptosis , Genetics , Cell Cycle , Genetics , Cell Proliferation , Cyclin-Dependent Kinase Inhibitor p16 , Genetics , Gene Expression , Genes, p53 , Genetic Vectors , HL-60 Cells , K562 Cells , Plasmids , TransfectionABSTRACT
The objective was to explore the feasibility of differentiation of human umbilical cord blood mononuclear cells into endothelial cells induced by cytokines in vitro and to study the possibility of using cord blood stem cells in ischemic diseases therapy. The cells were isolated from umbilical cord blood by using lymphocyte separation solution, and committedly differentiated by using VEGF, bFGF and IGF-I in a liquid culture system. The results showed that the combination of cytokines produced a large number of caudated adherent cells and flow cytometric analysis revealed endothelial marker vWF expressed in about 80% cells, and the endothelial -specific Weibel-Palade body was detected in the cytoplasm by electronic microscope. It is concluded that human umbilical cord blood mononuclear cells may be induced to differentiate into endothelial cells induced by VEGF, bFGF and IGF-I. Human umbilical cord blood MNC may be an ideal source of adult stem cells for the treatment of the ischemic disease.
Subject(s)
Humans , Cell Differentiation , Cells, Cultured , Endothelial Cells , Cell Biology , Fetal Blood , Cell Biology , Fibroblast Growth Factor 2 , Pharmacology , Flow Cytometry , Insulin-Like Growth Factor I , Pharmacology , Leukocytes, Mononuclear , Cell Biology , Microscopy, Electron , Vascular Endothelial Growth Factor A , PharmacologyABSTRACT
<p><b>AIM</b>To explore whether the oligonucleotide uptake in hematological tumor cells is related to cellular species and proliferation.</p><p><b>METHODS</b>Intracellular mean fluorescence intensity was measured by flow cytometry.</p><p><b>RESULTS</b>After treatment with FITC-labeled G3139 at the concentration of 0.60 mumol.L-1 for 4 h, the G3139 uptake into peripheral blood mononuclear cell and bone marrow mononuclear cell in hematological tumor patients was significantly higher than that in normal control. There was different uptake of G3139 among the malignant hematological tumor cell strains, and the uptake in cells derived from monocyte, B lymphocyte and myeloid cell was much higher than that in cells derived from T lymphocyte. After treatment with all-trans retinoic acid (ATRA), HL60 cell proliferation was markedly inhibited and the uptake of G3139 decreased significantly.</p><p><b>CONCLUSION</b>Hematological tumor cells were capable of taking up oligonucleotide, and the oligonucleotide uptake in hematological tumor cells is related to its cellular species and its activation.</p>
Subject(s)
Humans , Biological Transport , Cell Division , Physiology , Genes, bcl-2 , Genetics , HL-60 Cells , Leukemia , Metabolism , Pathology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Metabolism , Pathology , Leukemia, Promyelocytic, Acute , Metabolism , Pathology , Leukocytes, Mononuclear , Metabolism , Lymphoma, Non-Hodgkin , Metabolism , Pathology , Oligonucleotides, Antisense , Metabolism , Thionucleotides , Metabolism , Tretinoin , Pharmacology , Tumor Cells, CulturedABSTRACT
The tumor suppressor gene p53 and p16, both of which play an important role in inhibition of tumorigenesis, are homozygously deleted in human myeloid leukemia cell line K562. To explore the inhibition of K562 cell proliferation by wild type p16 and p53 genes, both p16 and p53 genes were co-transfected into K562 cells mediated by liposome. The expression of the two genes was measured by immunocytochemical method, the cell cycle was analysed by flow cytometry, and the number of recovered viable cells was assessed after transfection. After co-transfection, the p53 and p16 positive cells were 23% and 28%, respectively. The results showed that co-transfection of p16 and p53 genes significantly inhibits cell proliferation comparing with transfection either by p16 gene or by p53 gene (P < 0.05). Expression of p16 and p53 proteins increased the cell number in G(1) phase but decreased the cell number in S phase. It is concluded that co-transfection of p16 and p53 genes has a stronger growth-inhibitory effect on K562 cell growth than that of transfection only by p16 gene or by p53 gene, may be a pathway for gene therapy in leukemia.