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Decorin recombinant human leukemia K562 cells in vitro effects of experimental research

Author: JingGang
Tutor: WangGuiQin
School: Shanxi Medical
Course: Immunology
Keywords: Recombinant human decorin K562 cells Apoptosis Cell cycle BCL-XL Mcl-1 Bax Doxorubicin TGF-β1mRNA
CLC: R733.7
Type: Master's thesis
Year: 2010
Downloads: 19
Quote: 0
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Abstract


Purpose: The effects of recombinant human decorin (rhDCN) leukemia K562 cell growth inhibition, apoptosis, and to explore the possible mechanism of action. Method: 1. Would have been constructed pcDNA3.1 ()-DCN eukaryotic expression vector by EcoRI, NotI digestion, sequencing. The correct identification of a large number of recombinant plasmid purification amplification through LipofectamineTM2000 liposome-mediated transfection leukemia K562 cells in logarithmic growth phase extraction of total RNA 48 h after transfection cells by RT-PCR. The test is divided into a 0.9% NaCl solution group, pcDNA3.1 () / of K562 group of pcDNA3.1 () -DCN/K562 group and liposome group. 4 by Reiter staining the cell morphology after transfection, MTT assay to detect cell proliferation activity, propidium iodide (PI) staining, flow cytometry analysis of cell cycle and apoptosis. 5.Western blot detection of apoptosis protein BCL-XL, Mcl-l and Bax. Results: 1 double digestion of recombinant plasmid, match the size of the resulting gene fragment with the expected gene fragment; the DCN gene sequence in the sequencing results with Genebank entirely consistent, no mutation. 2. PcDNA3.1 ()-DCN recombinant plasmids were transformed into K562 cells by liposome transfection method, RT-PCR results confirmed successful transfection. 3.pcDNA3.1 () -DCN/K562 group Reiter staining showed typical apoptotic morphological changes, other groups no obvious apoptotic morphological changes. 4.MTT results show pcDNA3.1 () -DCN/K562 cell proliferation inhibition rate (transfected 24h 16 ± 1.08% of transfected 48h 14 ± 1.01%, transfection 72h to 20 ± 1.19%) was significantly higher proliferation inhibition rate at the corresponding point in time other groups, the difference was statistically significant (P lt; 0.05); the FCM test results, pcDNA3.1 () -DCN/K562 group apoptosis rate (20.15 ± 1.31%), the cells in the G0 / G1 phase cell percentage (51.15 ± 0.57%) compared with other groups results increased significantly, and the difference was statistically significant (P lt; 0.05). The pcDNA3.1 5.Western blot results show () -DCN/K562 group and the other groups of BCL-XL, Mcl-1 protein expression to reduce elevated Bax protein expression. Conclusion: rhDCN recombinant plasmid can effectively inhibit the proliferation of K562 cells and induced apoptosis. rhDCN of the cell cycle and apoptosis-related protein BCL-XL, Mcl-1, Bax impact may play a role in the mechanism and provide new experimental evidence for the biological treatment of leukemia. Objective: To study recombinant human decorin (rhDCN) Joint doxorubicin (ADM) on leukemia K562 cell growth, and analyze the possible mechanisms. Methods: K562 cells cultured outside the the RPMI1640 culture liquid containing 10% fetal bovine serum, liposome-mediated transfection of K562 cells in logarithmic growth phase, test is divided into 0.9% NaCl solution group, pcDNA3.1 ()-DCN / K562 group the, ADM/K562 group, pcDNA3.1 ()-DCN the plus ADM/K562 group. Reiter staining morphological changes; detected by MTT cell proliferation activity; flow cytometry (FCM) Annexinv / PI double staining analysis of apoptosis; using RT-PCR analysis of each treatment group K562 cells the TGF-β1mRNA level expression changes. Results: pcDNA3.1 ()-DCN plus ADM/K562 group of cells than separate the DCN and ADM cells, staining showed more pronounced morphological changes of apoptosis; MTT results showed that the inhibition of cell proliferation rate of the combination group (61 ± 1.32%) significantly the FCM test results show that the combination group apoptosis rate (61.30 ± 0.9%) higher than the intervention group (DCN group 20 ± 1.9%; the ADM group of 47 ± 1.04%) (P lt; 0.05), with separate intervention group (DCN group 28.25 ± 1.3%; ADM group 31.85 ± 1.5%) and relatively increased significantly (P lt; 0.05), RT-PCR results showed that the decrease in joint cells TGF-β1 mRNA transcription. Conclusion: rhDCN can significantly enhance the killing effect of ADM on K562 cells, increase the rate of apoptosis of tumor cells. TGF-β1 mRNA transcription synergy, its specific mechanism needs further research.

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CLC: > Medicine, health > Oncology > Hematopoietic and lymphoid neoplasms > Leukemia
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