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Study of the Antitumor Potency of P16 Gene in Osteosarcoma U-2OS Cells

Author: XiaZuoFan
Tutor: ZhangJian
School: Chongqing Medical University
Course: Surgery
Keywords: p16 Osteosarcoma U-2OS Cell Proliferation Cell Cycle Apoptosis
CLC: R738.1
Type: Master's thesis
Year: 2011
Downloads: 11
Quote: 0
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Abstract


Objective: Construction and identification of the p16 gene eukaryotic expression plasmid pcDNA3 - HA- p16 inhibitory effect of exogenous p16 gene on the osteosarcoma cell line U - 2OS . Methods : RT - PCR amplification of the p16 gene . By molecular biology techniques to build p16 eukaryotic expression plasmid pcDNA3 - HA - p16 . Liposomal transfection method exogenous p16 gene into cell U - 2OS . 4 by immunofluorescence , Western blot was used to verify the expression of pcDNA3 - HA - p16 in the U - 2OS cells . 5 CCK-8 assay cell proliferation . 6. Application PI staining analysis of cell cycle distribution . 7 DNA ladder method combined with PI staining of apoptotic cells was observed . Results: 1 by restriction enzyme digestion and DNA sequencing of the recombinant plasmid was successfully constructed the p16 gene eukaryotic expression plasmid pcDNA3 - HA- p16 recombinant plasmid by lipofection in osteosarcoma cell line U- the stable expression 2OS , p16 is mainly distributed in the nucleus . P16 significantly inhibited the proliferation of U-2OS ( P lt ; 0.05 ) , showed significant dose-effect relationship and this effect within a certain range . P16 causes the U - 2OS G1 phase cells was significantly higher ( P lt ; 0.05 ) , while the proportion of cells in S phase was significantly lower ( P lt ; 0.05 ) P16 resulted in the U - 2OS apoptosis rate increased significantly ( P lt ; 0.05 ) DNA after agarose gel electrophoresis showed a typical DNA ladder . Conclusion: 1 . Successfully constructed p16 eukaryotic expression plasmid pcDNA3 - HA - p16 the liposomal transfection stable expression in osteosarcoma cell line U - 2OS . (2) tumor suppressor gene p16 significantly inhibited the proliferation of U - 2OS cells and this effect is shown clear dose-response relationship within a certain range ; p16 led to the U - 2OS cell cycle arrest in G1 phase ; promote apoptosis and significantly . Thus p16 significantly inhibited the effect of the U-2OS cells .

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