| Abstrakt: |
Islet cell lines were produced by retroviral transduction of SV-40 T antigen to monolayer cultures of neonatal rat islets. One line, RN0-11, showed evolution of phenotypes in vitro. It evolved from a non-hormone secreting cell line to an insulin secreting line. It further developed glucagon producing capability before it lost all hormone producing phenotypes. At passage 8, RN0-11 cells secreted a small amount of insulin 25 ng/10(6) cells/24 hours. They were unresponsive to glucose and secreted 1.7-1.9 ng of insulin/10(6) cells/2 hours under various levels of glucose. At passage 16, they secreted 2,577 ng of insulin/10(6) cells/24 hours and responded to glucose stimulation in static incubation. The insulin secreted by these cells at 0, 2.8, 5.5, 11.1, 16.7, and 27.7 mM of glucose was 6.46 +/- 2.56, 17.74 +/- 2.66, 32.24 +/- 0.58, 30.66 +/- 1.59, 33.55 +/- 4.83, and 20.95 +/- 2.17 ng/10(6) cells/2 hours respectively. The responsiveness to glucose and the ability to secrete insulin diminished as cells were passaged in culture, and by passage 35 no insulin was detectable in medium under any level of glucose tested. Northern blot analyses also showed corresponding changes of insulin expression in these cells at different passages. In addition, glucagon was detectable at passage 14 by immunocytochemistry and at passage 16 by Northern blot analysis. By passage 35, no insulin or glucagon expression was detectable by Northern blot analysis or immunocytochemistry. Immunocytochemical staining of these cells at passage 14 showed insulin-positive and glucagon-positive cells and cells positive for both insulin and glucagon. Presence of insulin and glucagon in the same cells suggests single clonality of the cell line. The evolution of RN0-11 cells in vitro provides an opportunity to study the development of islet cells. |
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