Identification of genes regulating the differentiation of adrenocotical stem cells and their functional mechanisms

哺乳动物肾上腺由球状带(Zg)、束状带(ZF)和网状带(ZR)三个区组成。尽管三个区域的细胞来自相同的来源，即中胚层来源，但每个区域具有不同的功能。到目前为止，肾上腺核的干细胞和/或前体细胞尚未被鉴定，肾上腺核的分化、形成和维持的分子机制尚不清楚。我们之前在大鼠的ZG和ZF之间发现了一个功能未分化的细胞区(ZU)。ZU内及周围皮质中有丰富的复制细胞，提示ZU含有干细胞，可分化为ZG或ZF/ZR细胞。为了建立分析分化过程的体外模型系统，我们试图获得与ZU细胞类似的处于未分化阶段的肾上腺皮质细胞系。从含有SV40大T抗原基因温度敏感突变体的转基因小鼠的肾上腺原代培养中建立了具有这种特性的细胞系。然后，我们试图通过差异杂交筛选的方法克隆在未分化细胞系中特异表达的编码蛋白的cDNA。获得了多个克隆。其中一个克隆可能编码一种新的细胞外基质蛋白。另一个基因编码了一个新的蛋白质，其氨基酸序列没有任何已知的同源序列。在ZG-ZU和ZU-ZF区域分别检测到它们的mRNAs。它们的表达部位对应于肾上腺皮质细胞增殖和分化最活跃的区域。这些结果表明，新发现的在未分化细胞系中特异表达的蛋白质在肾上腺皮质细胞的分化和肾上腺皮质的形成中起作用。

Mammalian adrenal codex is composed of three zones, the zona glomerulosa (zG), zona fasciculata (zF), and zona reticularis (zR). Each zone has a different function, though cells in three zones are of the same origin, I.e. mesodermal origin. So far, stem cells and/or projenitor cells of the adrenal corex have not been identified, and molecular mechanisms of the differentiation, formation and maintenance of adrenal corex have remained obscure. We previously found a functionally undifferentiated cell zone (zU) between zG and zF in rats. Replicating cells in the cortex were abundant in and around zU, suggesting that zU contains stem cells which are capable to differentiate into zG or zF/zR cells. To develop in vitro model systems for analyzing the differentiation processes, we attempted to obtain adrenocodical cell lines at the undifferentiated stage similar to that of zU cells. Cell lines with such properties were established from a primary culture of adrenal glands from transgenic mice harboring a temperature sensitive mutant of SV40 large T antigen gene. Then, we tried to clone cDNAs encoding proteins which were expressed specifically in the undifferentiated cell lines with the aid of differential hybridization screening. Several cDNA clones were isolated. One of the cDNA clones was found to presumably encode a new extracellular matrix protein. Another cDNA encoded a new protein whose amino acid sequence did not have any known homologous sequence. Their mRNAs were detected in the regions from zG to zU and from zU to zF, respectively. The sites of their expression corresponded to the regions where cell proliferation and differentiation of the adrenocodical cells took place most actively.These results suggest that the newly discovered proteins expressing specifically in the undifferentiated cell lines have roles in the differentiation of adrenocortical cells and also in the formation of the adrenal cortex.