The induction of the differentiation into vascular smooth muscle cells and cardiomyocytes by the transcription factor Mrf2 (Modulator Recognition Factor 2)

建立了血管平滑肌细胞的体外分化体系。利用该系统，我们克隆了一个新的转录因子，Mrf 2（调制识别因子2）作为平滑肌细胞分化的重要关键分子。本研究主要研究Mrf2.1的作用。Mrf 2的表达利用大肠杆菌制备的Mrf 2蛋白的半部分制备了兔多克隆抗体。用此抗体，我们可以观察到Mrf 2蛋白的核定位，它是通过表达载体转染到293 T细胞，但它似乎没有工作的免疫组织化学。接下来，我们使用由Mrf 2氨基酸序列设计的特异性寡肽制备了另外三种抗体。其中一种只能检测Mrf 2的β-亚型，而其他两种都可以检测α和β亚型。此外，我们制备了用于原位杂交的构建体。2、Mrf 2对平滑肌细胞特异性启动子的调控为了检测Mrf 2是否能调控平滑肌细胞特异性启动子，我们制备了在其启动子区含有CArG盒或SM 22 α等启动子的荧光素酶构建体，并进行了启动子分析。但是，这些启动子的活性不受Mrf 2表达的影响。Mrf 2和SRF的共表达不能调节启动子的活性。3、Mrf 2病毒载体的构建我们已经构建了Mrf 2的逆转录病毒和腺病毒载体。最近，Mrf 2在脂肪细胞分化中的作用被报道。我们对Mrf 2在平滑肌细胞和脂肪细胞分化过程中的作用非常感兴趣。

We established the in vitro differentiation system of vascular smooth muscle cells. Using this system, we cloned a novel transcription factor, Mrf2 (Modulator Recognition Factor 2) as an important key molecule for the differentiation of the smooth muscle cells. Our research has focused on the role of Mrf2.1, The expression of Mrf2We made a rabbit polyclonal antibody using the half portion of the Mrf2 protein, which was prepared using the E.Coli. With this antibody, we could observe the nuclear localization of Mrf2 protein, which is transfected into 293T cells by the expression vector, but it did not seem to work for the immunohistochemistry. Next, we made additional three antibodies using the specific oligopeptides designed from the Mrf2 amino acid sequence. The one would detect only beta-isoform of Mrf2, and the others should detect both alpha and beta isoforms. Moreover, we prepared the constructs for in situ hybridization. We are now analysing the expression pattern of Mrf2 from the both results.2, The regulation of the smooth muscle cell specific promoters by Mrf2To test if Mrf2 can regulate smooth muscle cell specific promoters, we prepared luciferase constructs with the CArG box, or SM22 α promoter et al. in their promoter regions, and performed the promoter assays. But, the activities of these promoters were not affected by Mrf2 expression. The co-expression of Mrf2 and SRF could not regulate the promoter activity. We made several deletion mutants of Mrf2 and are now testing if they could change the promoter activities, as dominant negative or positive mutants.3, The construction of the virus vectors of Mrf2We have constructed the retro-virus and adeno-virus constructs of Mrf2.Recently, the role of Mrf2 in the differentiation of adepocytes was reported. We are very interested in the role of Mrf2 during the differentiation of both smooth muscle cells and adepocytes.