Human β-cell gene profiling is a powerful tool for understanding β-cell biology in normal and pathological conditions. Assessment is complicated when isolated islets are studied because of contamination by non-β-cells and the trauma of the isolation procedure. Objective: The objective was to use laser capture microdissection (LCM) of human β-cells from pancreases of cadaver donors and compare their gene expression with that of handpicked isolated islets. Design: Endogenous autofluorescence of β-cells facilitated procurement of purified β-cell tissue from frozen pancreatic sections with LCM. Gene expression profiles of three microdissected β-cell samples and three isolated islet preparations were obtained. The array data were normalized using DNA-Chip Analyzer software (Harvard School of Public Health, Boston, MA), and the lower confidence bound evaluated differentially expressed genes. Real-time PCR was performed on selected acinar genes and on the duct cell markers, carbonic anhydrase II and keratin 19. Results: Endogenous autofluorescence facilitates the microdissection of β-cell rich tissue in human pancreas. When compared with array profiles of purified β-cell tissue, with lower confidence bound set at 1.2, there were 4560 genes up-regulated and 1226 genes down-regulated in the isolated islets. Among the genes up-regulated in isolated islets were pancreatic acinar and duct genes, chemokine genes, and genes associated with hypoxia, apoptosis, and stress. Quantitative RT-PCR confirmed the differential expression of acinar gene transcripts and the duct marker carbonic anhydrase II in isolated islets. Conclusion: LCM makes it possible to obtain β-cell enriched tissue from human pancreas sections without the trauma and ischemia of islet isolation.
Gene expression of purified beta-cell tissue obtained from human pancreas with laser capture microdissection.
MARSELLI, LORELLA;
2008-01-01
Abstract
Human β-cell gene profiling is a powerful tool for understanding β-cell biology in normal and pathological conditions. Assessment is complicated when isolated islets are studied because of contamination by non-β-cells and the trauma of the isolation procedure. Objective: The objective was to use laser capture microdissection (LCM) of human β-cells from pancreases of cadaver donors and compare their gene expression with that of handpicked isolated islets. Design: Endogenous autofluorescence of β-cells facilitated procurement of purified β-cell tissue from frozen pancreatic sections with LCM. Gene expression profiles of three microdissected β-cell samples and three isolated islet preparations were obtained. The array data were normalized using DNA-Chip Analyzer software (Harvard School of Public Health, Boston, MA), and the lower confidence bound evaluated differentially expressed genes. Real-time PCR was performed on selected acinar genes and on the duct cell markers, carbonic anhydrase II and keratin 19. Results: Endogenous autofluorescence facilitates the microdissection of β-cell rich tissue in human pancreas. When compared with array profiles of purified β-cell tissue, with lower confidence bound set at 1.2, there were 4560 genes up-regulated and 1226 genes down-regulated in the isolated islets. Among the genes up-regulated in isolated islets were pancreatic acinar and duct genes, chemokine genes, and genes associated with hypoxia, apoptosis, and stress. Quantitative RT-PCR confirmed the differential expression of acinar gene transcripts and the duct marker carbonic anhydrase II in isolated islets. Conclusion: LCM makes it possible to obtain β-cell enriched tissue from human pancreas sections without the trauma and ischemia of islet isolation.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
