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Inhibitory Effect of Adenovirus-mediated Mouse Endostatin on Lung Metastasis of Osteosarcoma-bearing Nude Mice

Author: MaXiaoHui
Tutor: CaiXuanSong；MeiJiong
School: Tongji University
Course: Surgery
Keywords: Cell lines Nude mice Adenovirus vector Osteosarcoma Lung metastases Endostatin Antiangiogenic The immune enzyme-linked adsorption reaction
CLC: R738
Type: Master's thesis
Year: 2006
Downloads: 29
Quote: 0
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Abstract


Objective: mouse endostatin adenovirus mediated through their own building and observed in vivo and in vitro expression and inhibition of tumor vasculature and Dutch osteosarcoma nude mouse lung metastasis, to explore the level of expression of endostatin with osteosarcoma lung metastasis. Methods: 1 to mEndostatin plasmid as a template by PCR method amplification recycling mEndostatin genes connection the mEndostatin gene adenovirus vector PDC315 build PDC315 a mEndo expression plasmid. This expression plasmid the the adenovirus recombinant plasmid pBGHE3 through lipofectamine 2000 transfection 293 cells by homologous recombinant adenovirus Ad-mEndo was purified using cesium chloride density gradient ultracentrifugation, 50% tissue culture infectious dose method The viral titer was determined. Osteosarcoma cell line MG-63, in vitro transfection using western blot detection of infection osteosarcoma of cell supernatant mEndostatin the expression and activity observed inhibition of angiogenesis chick chorioallantoic membrane assay (CAM). Establish an animal model of nude mice bearing human: osteosarcoma cell line MG-63, 0.2ml cell suspension (approximately 1 × 10 6 cells) / 30 injected into nude mice right forelimb The subcutaneous, after a week All tumor were randomly divided into three groups: the Ad-mEndo was group (10), Ad-EGFP group (10), PBS (10). In addition to unvaccinated tumor cells in nude mice (10) for the control group. Then each group a week respectively corresponding drug injection 200ul / five times in succession, the last blood with the immune enzyme-linked adsorption reaction (ELISA) to detect the expression levels of endostatin; measuring the long and short diameters of the tumor tumor volume was calculated with a vernier caliper and were killed after seven weeks to observe whether the lung metastases. Results amplified mEndostatin by restriction endonuclease digestion, PCR amplification, DNA sequencing confirmed murine endostatin recombinant adenovirus Ad-mEndo titer of 2.5 × 10 10 pfu / ml, in vitro efficiently transfected osteosarcoma cell line the secretory expression to inhibit angiogenesis activity mEndostatin. 2 treatment after the Ad-mEndo group tumor volume was significantly less than the Ad-EGFP and PBS group endostatin expression level was significantly higher than that of Ad-EGFP and PBS group, and there is a significant difference (P <0.05). While observing the lungs of three groups of nude mice found that the The lungs of the Ad-mEndo group found no tumor metastases, while the other two groups of nude mice lungs visible to a large number of scattered metastases, lung metastasis rate of> 70%. Conclusion: mouse endostatin adenovirus-mediated gene in the body, outside are highly expressed in significant inhibition of tumor angiogenesis and Dutch osteosarcoma lung metastases in nude mice. Endostatin expression level of lung metastasis has a direct relationship.

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