Analysis of Phenotypic Variation of Mast Cells by Using Messenger RNA Expression as a Marker

蛋白酶细胞的嗜碱性颗粒含有多种肥大细胞特异性蛋白酶。许多编码肥大细胞特异性蛋白酶的基因已经在小鼠中克隆出来。小鼠肥大细胞蛋白酶（MMCP） 1 ~ 5为乳糜酶，MMCP-6和-7为胰蛋白酶。采用原位杂交技术研究了影响肥大细胞特异性蛋白酶表达的因素。MMCP的表达模式受小鼠训练、肥大细胞分化组织和细胞因子受体信号的影响。1994年，我们通过培养存在T细胞源性细胞因子的小鼠骨髓细胞获得了培养肥大细胞（cmc）。从cmc中提取总RNA，得到MMCP-2、MMCP-4、MMCP-5、MMCP-6和肥大细胞羧肽酶的cdna。将cmc离心后包埋于石蜡中，使用地高辛标记的RNA探针进行原位杂交。由于技术问题在1994年得到解决，我们于1995年研究了肥大细胞特异性蛋白酶在不同培养条件下mRNA表达谱的变化。我们发现了肥大细胞最重要的生长因子干细胞因子受体c-kit受体酪氨酸激酶的功能获得突变。我们研究了通过c-kit受体的信号对肥大细胞特异性蛋白酶表达的影响。将组成型活化的c-kit cDNA导入IC-2肥大细胞系后，MMCP-6的表达增强。

Basophilic granules of mase cells contain various mast cell-specific proteases. Many genes encoding mast cell-specific proteases have been cloned in mice. Mouse mast cells proteases (MMCP) 1 to 5 are chymases, and MMCP-6 and -7 are tryptases. We investigated the factors which influenced the expression of mast cell-specific proteases by using in situ hybridization. The pattern of MMCP expression was affected by mouse atrains, tissues in which mast cells differentiated and signals from cytokine receptors. In 1994, we obtained cultured mast cells (CMCs) by cultureing bone marrow cells of mice in the presecnce of T cell-derived cytokines. Total RNA was extracted from CMCs, and cDNAs of MMCP-2, MMCP-4, MMCP-5, MMCP-6 and mast cell carboxypeptidase were obtained. CMCs were centrifuged and embedded in paraffin, and in situ hybridiazation using digoxigenin-labeled RNA probes was carried out. Since the technical problems were resolved in 1994, we studid the change in mRNA expression patters of mast cell-specific proteases under various culture condition in 1995. We found the gain-of-function mutations of c-kit receptor tyrosine kinase, which is the receptor for stem cell factor, the most important growth factor of mast cells. We investifated the effect of signals through the c-kit receptor on the expression of mast cell-specific proteases. Introduction of the constitutively activated c-kit cDNA into the IC-2 mast cell line resulted in the enhanced expression of MMCP-6.