Identification of the genes involved in the cell differentiation of the pancreas β cell and pre-clinical study of the diabetes model animal.

本实验的目的是寻找与肥大岛相关的基因，并检测这些基因mRNA的组织学定位。建立了一种高通量的原位杂交技术，并对KKAy和BALB/cA小鼠胰腺组织进行了双向差减文库的构建。从KK-BALB文库中筛选出162个克隆（K-B），从BALB-KK文库中筛选出153个克隆（B-K）作为特异表达克隆。经同源性分析和聚类分析，K-B中有29个基因，B-K中有23个基因。接下来，进行定量RT-PCR分析以选择克隆。最终筛选出11个KKAy特异性基因（包括1个未知基因）和10个BALB特异性基因（包括2个未知基因）。参与再生的Reg I、Reg II和Reg III β在KKAy小鼠中特异性增强其表达。在10周龄BALB胰腺中未检测到Reg基因。所有Reg基因仅在10周龄KKAy胰腺的腺泡细胞中表达，而在胰岛和外分泌管中不表达。而在27周龄的BALB胰腺腺泡细胞中，Reg I基因表达较强。Reg II基因在腺泡细胞中微弱表达。Reg III beta几乎检测不到。所有Reg基因仅在30周龄KKAy的腺泡细胞中强烈表达。有趣的是，胰岛周围细胞中Reg III β的表达减少。3.还在链脲佐菌素诱导的I型糖尿病小鼠中检查了KKAy特异性基因的表达。结果表明，Reg基因在KKAy胰岛β细胞再生过程中可能起重要作用。

The purpose of this experiment is to search the genes, which are involved in the hypertropic Island, and to examine the histological localization of the mRNA of these genes.As results, 1. We developed a novel high throughput in situ hybridization system to analyze the gene expression profiles steadily.2.The two-way subtraction libraries were made between the pancreas of 10 week-old KKAy and one of age matched BALB/cA. We selected 162 clones from KK-BALB library (K-B) and 153 from BALB-KK library (B-K) as specifically expressed clones. After homology analysis and clustering, 29 genes in K-B and 23 in B-K were identified. Next, quantitative RT-PCR analysis was done to select the clones. Finally, 11 KKAy specific including 1 unknown gene and 10 BALB specific genes including 2 unknown genes were selected. Reg I, Reg II and Reg III beta, which are involved in regeneration, were specifically enhanced their expression in the KKAy mouse. None of Reg genes was detected in 10 wks old BALB pancreas. All Reg genes were expressed only in acinar cells of 10 wks old KKAy pancreas, but not in the islet and the exocrine duct. However, in acinar cells of 27 wks old BALB pancreas, Reg I gene was strongly expressed. Reg II gene was faintly expressed in acinar cells. Reg III beta was hardly detectable. All Reg genes were strongly expressed only in acinar cells of 30 wks old KKAy. Of interest, the expression of Reg III beta was decreased in the cells surrounding the islet.3.Expression of KKAy specific genes were also examined in Streptozotocin induced type I diabetic mouse. As result, the expression of these genes was not specific in this model mouse.In conclusion, these data suggest that Reg genes may play important role in the beta cell regeneration in KKAy islet.