Isolating Human Islets of Langerhans Causes Loss of Decay Accelerating Factor (CD55) on -Cells
Abstract:It has previously been reported that human decay accelerating factor (DAF; CD55) is not expressed on cells isolated from human islets. We have investigated if this absence is caused by the islet isolation procedure and/or the single cell isolation technique. We focused on loss of DAF expression on -cells within the intact islet and on isolated individual -cells. We established that DAF was expressed in islets and on -cells prior to isolation by in situ analysis in the intact pancreas. In situ immunohistochemistry (IHC) was used to examine DAF expression on human pancreatic islets and isolated islets. A reverse transcriptase-polymerase chain reaction (RT-PCR) specific for human DAF mRNA was developed to measure mRNA levels in situ in islets within the intact pancreas, isolated islets, and purified -cells. -Cells were purified by fluorescence-activated cell sorting. DAF protein expression on these purified cells was measured using flow cytometry. Expression of DAF protein was present on the islets, including -cells within the human pancreas; however, comparative data from IHC and flow cytometry revealed the absence of DAF protein on -cells in both isolated islets and single cell preparations. Furthermore, compared to mRNA levels detected by in situ RT-PCR in the intact pancreas and in human HEK 293 cells, isolated islets, and purified human -cells showed downregulation of DAF mRNA. mRNA was detectable in both of these preparations by RT-PCR; levels were lower following both the islet isolation process (53%) and single cell preparation (a further 62%) compared to HEK 293 controls. Human islet allotransplantation might be more successful if either de novo transfer of DAF onto the isolated islets or novel techniques for islet isolation preserving DAF could be developed.
Document Type: Research Article
Publication date: 2008-12-01
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