| Popis: |
The use of a highly sensitive method of in situ hybridization capable of detecting one copy of interferon (IFN) mRNA per cell coupled with quantitative analysis of cytokine mRNA showed that the number of copies of mRNA per cell was directly proportional to the logarithm of the number of silver grains formed over that cell. More than 90% of both virus-induced human Namalwa and mouse C243 cells exhibited grain counts significantly greater than background values following in situ hybridization with riboprobes complementary to human IFN- alpha and mouse IFN- beta mRNA, respectively. Labeling was shown to be specific, as the labeled probe was displaced by a 200-fold excess of the specific unlabeled probe but not by a 200-fold excess of an unrelated probe. Although the large majority of cells within a population responded to induction, considerable variation was observed, however, in the content of IFN mRNA per cell: 24% of induced C243 cells contained more than 50 copies of IFN-beta mRNA per cell while 60% of the cells contained 10 copies or less. Low levels of IFN mRNA were also detected in both uninduced C243 cells and uninduced Namalwa cells. Five to 10% of peripheral blood mononuclear cells from normal donors expressed INF-alpha mRNA following induction in vitro. Approximately 1% of untreated peripheral blood mononuclear cells also exhibited low levels of IFN-alpha mRNA. Analysis of interleukin-6 (IL-6) mRNA showed that 97% of TNF-induced human MG63 cells contained IL-6 mRNA, although, again, the amount varied considerably from cell to cell. |
