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G1 phase of the cell cycle regulatory molecules in leukemia cells , lymphoma cells , and CD34 ~ cell proliferation , differentiation , apoptosis

Author: WangQinHong
Tutor: XieYi
School: Fudan University
Course: Internal Medicine
Keywords: Cell cycle Cyclin D Cyelin E p27 Cell differentiation Apoptosis Leukemia cells Gene transfection RNAi CD34 ~ cells
CLC: R733
Type: PhD thesis
Year: 2004
Downloads: 207
Quote: 0
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Abstract


The characteristics of the tumor cells differ from normal cells in its constant self-replicating, stubborn inverse differentiation and apoptosis. Uncontrolled cell proliferation is an important part of tumor, the disorder is closely related to cell cycle regulation. From the perspective of cell cycle regulation, we explore the G1 phase of the important regulatory molecules in tumor cells of the blood system, and hematopoietic stem / progenitor cell proliferation, differentiation, apoptosis. The first part of this paper, we the hexamethylene bisacetamide (HMBA) in vitro treatment of HL-60 and U937 cells to establish tumor cell proliferation, differentiation, apoptosis model to study the expression of Cyclin D, Cyclin E and p27 and related genes c-myc , Rb, Bcl-2 expression in the cell model change and its significance. The results show that, HMBA intervention in HL-60, after 72 hours of U937 cells, CD11b expression was significantly higher (such as 4mMHMBA treated CD11b amounted to 69.90 ± 5.02%, 46.41 ± 7.80%); The high dose HMBA promote expression of Annexin-V a (4mMHMBA increase group were 8.90 ± 0.81%, 11.23 ± 5.57%); Cell cycle analysis of HL-60 and U937 cells arrested in G1 phase. Created cell proliferation, differentiation, and apoptosis model, we found that the expression of Cyclin D expression of p27 expression was significantly higher dose-dependent manner (4mM group of Cyclin D expression in HL-60 cells was 63.00 ± 13.55%, p27 67.65 ± 10.49%); Cyclin E expression was significantly down-regulated (such as 4mM Cyclin E expression in HL-60 cells was 1.33 ± 0.23%), also showed a dose-dependent manner. Related genes c-myc, Rb, Bcl-2 expression in the cell model has also undergone significant changes (such as HMBA enables HL-60 cells c-myc, Bcl-2 mRNA expression were down, Rb mRNA in raised ). The results suggest that of Cyclin E and p27 and related genes may be involved in leukemia cell proliferation, differentiation, and apoptosis mechanism, therefore trying to cut of Cyclin E raised p27 is likely to inhibit tumor cell proliferation and promote differentiation, apoptosis The new strategy. From the point of view of the cell cycle exploring cell proliferation, differentiation, and apoptosis mechanism of literature rarely reported. In the second part, the p27 gene transfection experiments further confirmed the p27 tumor cell proliferation, differentiation and apoptosis. Successfully constructed the eukaryotic expression vector of the p27 gene pEGFP-C1/hp27, this carrier and adenovirus vectors Adp27 respectively transfected / infected HL-60 and Raji cells. The electric shocks transfection efficiency (HL-60) to 31.9%: of AdLacZ. infection rate (HL-60 and Raji) were 40.3%, 32%; pEGFP-C1/hp27 RT-PCR showed that the p27 gene mRNA expression significantly. p27 gene into the HL-60 and Raji cells can be induced significant cell growth and proliferation inhibition; same time lead to a significant increase in the HL-60 cells CD11b (such as 72 hours, pEGFP-C1/hp27 transfection induced HL-60 CD11b expression of 27.9 ± 4.1%; Adp27 the infection complex mouth. medical school to Bobo dissertation was 33.8 ± 5.1%); Adp27 infected HL-60, Raji cause significant apoptosis (such as 72 hours AnnexinV 10 PI a rate of 46.9 Guests , 5%, 35.7 disabilities 11.2%). The partial results confirmed that p27 can significantly inhibit the proliferation of tumor cells and significantly promote differentiation, apoptosis, suggesting that p27 in cell proliferation, differentiation, apoptosis importance of tumor of the blood system for possible further application of p27 gene Gene therapy is the foundation. Have not been reported. The third part of the experiment, RNAi technology blocks gene expression at the core of the cell cycle progression CDKZ. CDKZ SIRNA plasmid (pBS 6/CDKZ) electric shock perforation, transfection of HL-60 cells, Rl'-PCR display CDKZ mRNA expression significantly reduced, and showing a time-dependent manner; CDKZ siRNA transfection, HL 60 significantly inhibited cell growth and proliferation; differentiation markers CDllb expression was significantly higher (24 hours, 48 ??hours and 72 hours, respectively, to 17. belly 2.3%, 30.2 ± 3.5%, 49.7 disabilities 11.6%) suggesting that the HL-60 cells tend grain / single lineages. Results that CDKZ downregulated involved in tumor cell proliferation, differentiation mechanism, which interfere with the proliferation of tumor cells from the cell cycle point of view, to promote their differentiation then provides another new way to achieve the purpose of the anti-tumor. At home and abroad have not been reported. The fourth part of this paper, we build it ourselves p27 SIKNA expression plasmid (P sileneer U6 1 .0/hP 27), sequencing confirmed that the complete sequence of the insert that was successfully constructed. This plasmid cord blood eD34 cells transfected to electric shock, the transfection efficiency of 27.1 3 1.4%; confirmed after transfection Rl'-peR the en34 cells p27 gene mRNA expression was significantly lowered, we built p27 siRNA plasmid ; the p27 SIRNA plasmid transfection the total number of CD34 cells was significantly higher than the empty plasmid group (6,9 days: 6.1 Guests 0.2 x 106/mlvs 3.7 Guests 0.5 X 106/ml, p lt; 0.05,7.6 disabilities o.sxlo6zmlvs4 l disabilities o.6Xlo6/ml p lt; 0.05), while retaining the CD34 marker positive rate was also significantly higher than the empty plasmid group (6,9 days: 84.5 Guests 6.1% Vs 48.4 Guests 3.9%, p lt ; 0.05,63.3 disabilities 3.2% Vs 28.7 Guests 5.4%, p lt; 0.05). The partial results that can cause interference to p27 SIRNA cord blood CD34 cells significantly amplified, without affecting their differentiation potential to open up new channels for cord blood CD34 cells, which is different from the previous application cytokine amplification CD34 cells can cause cell differentiation, will have great significance for improving the CD34 hematopoietic stem / progenitor cell transplantation performance. Have not been reported.

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