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Mrf2 (Modulator Recognition factor 2)，作为平滑肌细胞分化的重要关键分子。我们的研究重点是Mrf2.1的作用，Mrf2的表达，我们利用Mrf2蛋白的一半部分，用大肠杆菌制备了兔多克隆抗体。利用该抗体，我们可以观察到Mrf2蛋白的核定位，通过表达载体将其转染到293T细胞中，但在免疫组化上似乎不起作用。接下来，我们利用Mrf2氨基酸序列设计的特异性寡肽制备了另外三种抗体。其中一种只检测Mrf2的β -异构体，而其他两种应该同时检测α和β -异构体。此外，我们还制备了用于原位杂交的结构体。我们现在正在从这两个结果中分析Mrf2的表达模式。2、Mrf2对平滑肌细胞特异性启动子的调控为了验证Mrf2是否可以调控平滑肌细胞特异性启动子，我们在启动子区域用CArG盒或SM22 α启动子等制备了荧光素酶构建体，并进行了启动子分析。但是，这些启动子的活性不受Mrf2表达的影响。Mrf2和SRF的共表达不能调节启动子活性。我们制造了Mrf2的几个缺失突变体，现在正在测试它们是否可以改变启动子活性，作为显性阴性或阳性突变体。3、Mrf2病毒载体的构建我们构建了Mrf2的逆转录病毒和腺病毒构建体。最近，Mrf2在脂肪细胞分化中的作用被报道。我们对Mrf2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.