Isolation,Identification And Sequence Analysis Of Differential Expressed Genes Induced By IL-1β In NIT-1 Pancreatic β-Cells

Diabetes is a worldwide disease and is the third cause of disease-related deaths only next to the cardiovascular diseases and the cancers. Among the types of the disease, type 1 diabetes is an autoimmune disease characterized by mononuclear cell infiltration in the islets of Langerhans (insulitis) and selective destruction of the insulin producing β -cells. The destruction of the β-cells is the key problem in the research for pathogenesis of type 1 diabetes.The molecular events involved in the 3 -cells death process are complex and poorly understood. Cytokine such as IL-1β may play a crucial role in inducing this process. Some genes of β -cell are changed under the action of IL-1 β, which may reflect the destruction mechanism of the $ -cells at molecular level. Therefore, clarification of the genes related to the cytotoxic effect of IL-1β on the β -cells, may provide insight into the understanding of the molecular mechanism of type 1 diabetes and the searching for new drugs that are effective for the prevention and treatment of this diseases.Aim: To get insight into the mechanism of β-cells destruction at molecular, by isolating and identifmg the differentially expressed genes in the NIT-1 cells and exploring the function of the genes after the cells being treated with IL-1β.Method: NIT-1 cell, a β -cell line derived from the transgeneic NODmice was used as the cell model. The adequate IL-1β concentration was obtained by measuring insulin release under the treatment of the cells with a series of IL-1β concentration. The differentially expressed genes were isolated by RD-PCR technique and cloned by transforming them into the Escherichia coli JM109. The positive clones were confirmed by using RNase assay. Corresponding plasmids were subject to sequence. Lastly, the sequences were submitted to GeneBank for homogenous contrast analysis.Result: The concentration of IL-1β was fixed at 50U/ml for all the experiments. 86 genes fragments which showed obvious differential expression were obtained. Out of the 86 gene fragments, 41 positive gene fragments were identified with RNase assay. Among the positives, there were 0)14 new gene ESTs with the following access numbers: CB518127, CB518128, CB518129, CB518130, CB518131, CB518132, CB518133, CB518134, CB518135, CB518136, CB518137, CB518138, CB518139 and CB518140. (2)27 known gene fragments among which 13 have been reported to be related to the destruction of 3 -cells induced by IL-1 β , 9 gene fragments have not been reported to be related to the destruction of β -cells induced by IL-1β , and 5 with functions were unknown.Conclusion: New gene fragments and those relating to the destruction of P -cells were obtained by the RD-PCR. It was supposed that iNOS, Hrnoxl, SRP72, RRM1 and KIF13A could be used as targets for screening drugs to prevent and treat type 1 diabetes.