| Summary: | 碩士 === 長庚大學 === 醫學生物技術研究所 === 95 === Severe diseases or injury usually cause apoptosis of cells, which may release apoptotic bodies into circulation. These circulating apoptotic bodies thus contain valuable information (e.g. the specific protein or gene expression) of the original cells from which they were derived and can be useful for diagnostics. The aim of this study is to develop a convenient procedure to collect circulating apoptotic bodies in which disease-specific mRNA can be detected. First, magnetic beads coated with annexin A5, which bound phosphatidylserine on the surface of apoptotic cells, were tested for their capability of capturing apoptotic bodies. In an optimized buffer (HEPES-buffered saline containing 15 mM CaCl2), up to 60% of apoptotic bodies could be recovered by the magnetic beads from a suspension. Real-time reverse transcription-PCR further proved that specific gene expression can be detected quantitatively from the collected apoptotic bodies. Next, suitable conditions for blood sample preparation were tested. The result showed that blood collected in an EDTA-tube had much higher recovery rate of apoptotic bodies than that collected in a heparin-tube, suggesting that heparin might interfere either the binding of apoptotic bodies to annexin A5 or the subsequent assays. We applied this method to collect apoptotic bodies from cancer patients and found that CK19 mRNA can be detected in patients’ blood. In conclusion, we have developed a magnetic bead-based procedure for collection of circulating apoptotic bodies which might prove to be useful for disease diagnosis.
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